Apparatus and methods for analysing fluorescent particles

ABSTRACT

According to an embodiment of the invention, an apparatus to detect fluorescence from a sample is disclosed. The apparatus comprises a sample plane onto which the sample is arranged, an excitation light unit including at least a light source to illuminate the sample, and a detection unit comprising at least a detector having at least 100,000 active detection elements to detect a fluorescence signal from the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/123,560 filed Nov. 4, 2011, which is the U.S. National Phase ofPCT/DK2009/050278 filed Oct. 21, 2009, which claims priority of DanishPatent Application PA 2008 01464 filed Oct. 21, 2008.

FIELD OF THE INVENTION

The present invention relates to a particle analyzer. More particularly,the invention relates to a method and an apparatus for analysis ofparticles, such as biological particles.

BACKGROUND OF THE INVENTION

Many modern techniques of biochemistry and biotechnology are based onthe analysis of biological particles, e.g. cells. Several parametersconcerning the type or species of the particles, as well as the state ofthe particles, such as viability, are among parameters and propertiesthat are routinely investigated. Further information about intercellularstatus is also frequently determined. In this field, the method ofluminescence detection, e.g. fluorescence detection, has gained a wideapplication, mainly due to its inherent specificity and sensitivity.

Photoluminescent analysis of material, such as biological material, isbased on illuminating a sample with light (excitation) of a givenwavelength and collecting light emitted (emission) from the sample,parts or components of the sample, at another substantially differentwavelength. The difference in wavelength between excitation and emission(generally called Stoke's shift) is a property of the sample beinganalysed, more generally a property of the photoluminescent moleculespresent in the sample. If the Stoke's shift is great enough to allowsubstantial optical separation of excitation and emission light, it isfeasible to use the method of photoluminescence for the analysis of thematerial.

The photoluminescent emission (e.g. phosphoresce or fluorescence) istypically low in intensity compared to the excitation light, usually bythe order of several magnitudes. The fact that emission is detectedagainst “darkness”, makes the method well suited, since many of thedetectors commercially available show low response to “darkness” whileresponding considerably well to light, e.g. photons, striking thedetector. Nevertheless improved sensitivity, e.g. expressed as increasedemission, is typically a favourable property and therefore there existcurrently numerous methods for that purpose in the prior art.

Increase in the intensity of excitation light generally increases theamount of emitted light, since the probability of generating emission isproportional with the number of photons interacting withphotoluminescent molecules. One often used method for the increase ofexcitation intensity is the use of laser, which are available inconfigurations where the amount of emitted light is strong, both sincethe amount of emitted light, e.g. expressed as energy flux, isconsiderable, but also since the light from a laser is easily focusedonto a small area, thus generating high light density, e.g. expressed asemitted energy per area.

Another method for illumination of photoluminescent material is to use adispersed light source, such as a lamp or a light emitting diode. Theadvantage of such light sources is that they emit dispersed light, thusallowing the illumination of a considerably large area. One commondrawback to these light sources has typically been the relatively poordegree of homogeneous illumination of the sample material, obtainedsimultaneously with high degree of efficiency, defined by the fractionof emitted light striking the sample material.

Often preferred equipment for biological analyses is a microscope,typically equipped with two or more objectives for variablemagnifications. Further, fluorescence detection requires a wavelengthspecific excitation and emission filters. The operation of a microscopehas to some degree been automated, mainly through the implementation ofimage analysis. However, even with such automation, the operation of amicroscope is primarily a manual task, requiring considerable trainingof the operator.

Automated flow cytometers are also used for analyzing particles such ascells. Flow cytometer is a synonym for a wide range of equipmentcharacterised by analysing particles under flow conditions, where theseconditions usually allow individual particles to be analysed one at atime. Flow cytometer in certain versions make complex analyses ofbiological particles available, but flow cytometers are difficult to usebecause the operation usually requires considerable operator skill.

The apparatus and method of the present invention also addresses theareas of cell viability. The determination of cell viability isimportant for assessing the effects of e.g. drugs, environmentalpollutants, temperature, ionic extremes and radiation on cells andtissues. Cell membrane integrity is commonly used as indicator of cellviability. A feature of loss of membrane integrity is the formation ofpores that permit the passage of low molecular weight molecules (MW<2000Daltons) in and out of the cell. The enhanced permeability has been thebasis for many cell viability assays. The most common methods used forviability measurements are ⁵¹Cr release, Trypan blue exclusion and thecombination of different fluorescent dyes to detect live and dead cells.U.S. Pat. No. 6,403,378 describes a method based on membrane integritythat utilizes two fluorescent dyes, one which labels all dead cells withcompromised membranes while the other labels all living cells. To obtainreliable results for different cell populations and densities using atwo-dye method it is crucial to carefully control the amount of each dyeand the incubation time used to stain the cells. Propidium iodide andethidium bromide are excluded from the cytosol, and hence the nucleus,of viable cells and are mentioned as the most common fluorescent tracersfor staining dead cells. In contrast, acridine orange and Hoechst-33342are readily taken up by viable cells and are often used as fluorescentprobes for staining living cells.

Acridine orange (IUPAC name: N,N,N′,N′-tetramethylacridine-3,6-diamine,synonyms: Basic Orange 3RN, Euchrysine, Acridine Orange NO, RhodulinOrange NO, Waxoline Orange etc.) is a nucleic acid selective fluorescentcationic dye that interacts with DNA and RNA by intercalation orelectrostatic attractions. When bound to double stranded DNA and RNAacridine orange has excitation maximum at 502 nm and an emission maximumat 525 nm. When it associates with single stranded nucleic acid, theexcitation maximum shifts to 460 nm and the emission maximum shifts to650 nm. Acridine orange is also known to show alterations of absorbanceand fluorescence properties in its different forms. The monomeric dye insolution exhibits a green fluorescence, whereas the stacking of acridineorange in oligomeric structures will have a red emission (Kapuscinski etal., 1982: Luminescence of the solid complexes of acridine orange withRNA. Cytometry 2, pp. 201-211). This alteration results from aconcentration-dependent increase in resonance energy transfer amongindividual acridine orange molecules, and increasing concentrations ofacridine orange in a solution will induce progressive quenching of thegreen emmission (Minot et al., 1997: Characterization of Acidic Vesiclesin Multidrug-resistant and Sensitive Cancer Cells by Acridine OrangeStaining and Confocal Microspectrofluorometry. The Journal ofHistochemistry & Cytochemistry 45(9): pp. 1255-1264). Acridine orangewill also enter acidic compartments such as lysosomes and becomeprotonated and sequestered. In these low pH conditions, the dye willemit red light when exited by blue light. In conclusion, this shows thatthe fluorescence emission spectrum of acridine orange is affected bymany factors, including the gross secondary structure of thepolynucleotides, pH and the concentration of acridine orange.

DAPI or 4′,6-diamidino-2-phenylindole is another fluorescent dye thathas been described as cell permeable and useful for staining of livingcells (e.g. Betty I. Tarnowski; Francis G. Spinale; James H. Nicholson.1991. Biotechnic and Histochemistry, 66: 296-302). However, carefulstudies in our laboratories have revealed that DAPI penetrates cellswith a rather slow kinetics. Thus, by controlling the concentration andincubation time DAPI can be used as a probe for staining nonviable cellsand can therefore be used to discriminate between live and dead cells.DAPI preferentially binds to double stranded DNA and associates with ATclusters in the minor groove. When bound to double-stranded DNA itsabsorption maximum is at 358 nm and its emission maximum is at 461 nm.Binding of DAPI to DNA produces a 20-fold fluorescence enhancement. DAPIwill also associate to RNA, though in a different binding mode. Theemission peak of the DAPI/RNA complex is red-shifted to around 500 nmand the quantum yield is only 20% of that of the DAPI/DNA complex.

The combination of acridine orange and DAPI has not previously suggestedbeen as or demonstrated to be suitable for a simultaneous or two-colorfluorescence assay of cell viability.

The apparatus and method of the present invention also addresses theareas of transfection. Transfection, the introduction of foreign nucleicacid (DNA or RNA) into a eukaryotic cell, is a common and importantlaboratory procedure for studying the gene and protein function inliving cells. There are numerous methods available for cell transfectionsuch as formation of complexes of the nucleic acid with either DEAEdextran or calcium phosphate to allow cell uptake by endocytosis, oreletroporation, which employs pulses of voltage to form pores in theplasma membrane through which the nucleic acid can enter. Mosttransfection procedures, however, involve complexes of nucleic acids andcationic lipids followed by fusion with cells and delivery of DNA/RNAinto the cytosol. While rather routine, transfection requiresoptimization of assay conditions for different cell types. There are avariety of methods for determining transfection efficiency in cellpopulations. Most of these monitor the expression of a fluorescent,luminescent or colorimetric reporter gene. The reporter gene can bepresent on the same vector as the gene of interest or on a separatevector. Convenient reporter genes for measuring transfection efficiencyis autofluorescent proteins, e.g. green fluorescent protein (GFP)isolated from the jelly fish Aequorea victoria and red fluorescentprotein (RFP) developed from the marine anemone Discosoma striata, bothof which enable assays on living cells and requires no substrate forgeneration of fluorescence. When excited by blue light, GFP emits greenlight, whereas RFP emits orange/red light when excited with green light.Moreover, the combination of GFP with appropriate dyes allows multiplexanalysis to estimate e.g. viability, cytotoxity and apoptosis.

Another approach for monitoring transfection efficiency employsfluorescently labeled nucleic acids as reporter, e.g. Cy5-labeled siRNAto optimize RNAi silencing experiments.

In one method of analysis, the GFP transfected cells are incubated withDACM and propidium iodide (PI). DACM reacts with thiols, the level ofwhich is low in dying/dead cells, to produce a blue fluorescent productin living cells. In contrast, PI only penetrates cells with damagedmembranes and, thus, only labels DNA of dead cells. Cells labeled withDACM are detected by UV/violet excitation and measuring blue light,whereas PI labeled cells are detected by green light excitation andmeasuring the emitted red light. Cells expressing GFP (transfectedcells) are monitored by blue light excitation and measuring green light.Information about transfection efficiency and e.g. viability can beextracted from the data.

In another method of analysis, the RFP transfected cells are incubatedwith DACM and acrdine homodimer. Acridine homodimer only penetratescells with damaged membranes and, thus, only labels dead cells. Livingcells labeled with DACM are detected by UV/violet excitation andmeasuring blue light, whereas dead cells labeled with acridine homodimerare detected by blue light excitation and measuring the emitted greenlight. Cells expressing RFP (transfected cells) are monitored by greenlight excitation and measuring red light. Information about transfectionefficiency and e.g. viability can be obtained from the data.

In a third method of analysis, cells transfected with siRNA, labeledwith a green fluorophore, e.g. FITC, are incubated with DACM and PI.Living cells labeled with DACM are detected by UV/violet excitation andmeasuring blue light, whereas dead cells labeled with PI are detected bygreen light excitation and measuring the emitted red light. Cellsharboring siRNA (transfected cells) are monitored by blue lightexcitation and measuring green light. Information about transfectionefficiency and e.g. viability can be pulled out from the data.

The combination of a thiol-reacting reagent and a cell impermeable DNAstain in a cell population transfected with nucleic acid has notpreviously been suggested or demonstrated to be suitable for asimultaneous assay of transfection efficiency, cell viability andcytotoxity.

The apparatus and method of the invention also addresses the area ofcell cycle. The cell cycle represents the most fundamental and importantprocess in eucaryotic cells. An ordered set of events, culminating incell growth and division into two daughter cells, the cell cycle istightly regulated by defined temporal and spatial expression,localization and destruction of several cell cycle regulators. Cyclinsand cyclin-dependent kinases (CDK) are major control switches for thecell cycle, causing the cell to move from G₁ to S or G₂ to M phases. Ina given population, cells will be distributed among three major phasesof cell cycle: G₁/G₀ phase (one set of paired chromosomes per cell), Sphase (DNA synthesis with variable amount of DNA), and G/M phase (twosets of paired 2 chromosomes per cell, prior to cell division).

The most common approach to determine the cell cycle stage is based onmeasurement of cellular DNA content. DNA content can be determined usingfluorescent, DNA-selective stains that exhibit emission signalsproportional to DNA mass. Cellular fluorescence is measured by flow,image or laser scanning cytometry. This analysis is typically performedon permeabilized or fixed cells using a cell-impermeant nucleic acidstain, but is also possible using live cells and a cell-permeant nucleicacid stain.

Because cell cycle dysregulation is such a common occurrence inneoplasia, the opportunity to discover new targets for anticancer agentsand improved therapeutics has been the focus of intense interest. Thecell cycle assay has applicability to a variety of areas of life scienceresearch and drug development, including cancer biology, apoptosisanalysis, drug screening and measuring health status of cell cultures,e.g. in bioreactors.

DAPI is a competent dye for measurement of the cell cycle stage.However, excitation of DAPI requires a UV light source and standard flowcytometers usually come without a UV light source hampering the use ofDAPI.

The apparatus and method of the invention also addresses the area ofcell death. Cell death may occur by two distinct mechanisms, necrosis orapoptosis. Necrosis occurs when cells are exposed to harsh physical orchemical stress (e.g., hypothermia, hypoxia) while apoptosis is atightly controlled biochemical process by which cells are eliminated andwhere the cell is an active participant in its own termination(“cellular suicide”). Apoptosis is one of the main types of programmedcell death which occur in multicellular organisms and is characterizedby a series of events that lead to a variety of morphological changes,including blebbing, nuclear fragmentation, chromatin condensation, cellshrinkage, loss of membrane asymmetry and translocation of the membranephospholipid phosphatidylserine (PS) from the inner to the outer leafletof the plasma membrane.

Apoptosis is both a very complex and very important process anddysregulations in the apoptosis machinery may lead to very severediseases. A growing body of evidence suggests that resistance toapoptosis is a feature of most, if not all types of cancer. Moreover,defects in apoptosis signaling contribute to drug resistance of tumorcells. In the other hand may hyperactivity of the apoptotic processesalso cause diseases such as neurodegenerative diseases as seen inParkinson's or Alzheimer's Diseases, where apoptosis is thought toaccount for much of the cell death and the progressive loss of neurons.

As apoptosis play a very important role in a wide array of biologicalprocesses, including embryogenesis, ageing, and many diseases, this typeof programmed cell death is the subject for many studies, and tools foreasy detection and investigation of apoptosis are desirable.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to an apparatus todetect fluorescence from a sample, said apparatus comprising a sampleplane onto which the sample is arranged, an excitation light unitincluding at least a light source to illuminate the sample, and adetection unit comprising at least a detector having at least 100,000active detection elements to detect a fluorescence signal from thesample.

Another embodiment of the invention relates to an apparatus toilluminate a sample, said apparatus comprising a sample plane having anillumination region onto which the sample is arranged; an excitationunit having a light source to generate an excitation light; and anoptical system comprising a lens unit, having a micro lens array wherethe micro lens array comprises a plurality of lenses arranged in a twodimension arrangement, to receive the excitation light and generate anillumination light that is directed towards the illumination region;wherein, the lens unit produces a homogeneous illumination light to beprojected on the illumination region of the sample plane with a highillumination efficiency.

In one embodiment of the invention, a method for illuminating a sampleis explained. The method comprises of arranging a sample on a sampleplane having an illumination region of at least 0.5 mm²; generating anexcitation light using an excitation unit having a light source; andgenerating an illumination light, directed towards the illuminationregion, using a lens unit that comprises of a micro lens array having aplurality of lenses arranged in a two dimension arrangement; wherein,the lens unit produces a homogeneous illumination light to be projectedon the illumination region of the sample plane with a high illuminationefficiency.

In another embodiment of the present invention, a method for detectingfluorescence from a sample is described. The method comprises ofarranging a sample on a sample plane, illuminating the sample with anexcitation light using an excitation light unit having at least a lightsource, and detecting a fluorescence signal from the sample using adetection unit comprising at least a detector having at least 100,000active detection elements.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the invention, together with its advantages, may bebest understood from the following detailed description taken inconjunction with the accompanying figures in which:

FIGS. 1-A-1-C illustrate a light source for the illumination of a sampleand a prior art apparatus system for focusing the illumination lightonto sample material;

FIGS. 2-A-2-B illustrate a system for the illumination of a samplematerial using a micro lens array;

FIG. 3 illustrates the comparison of two methods for the illumination ofsample material;

FIGS. 4-A-4-B illustrate an implementation according to the presentinvention;

FIGS. 5-A-5-B illustrate a size of illumination region according to thepresent invention;

FIGS. 6-A-6-B illustrate a micro lens array element according to thepresent invention;

FIG. 7-A illustrates an arrangement of the apparatus where the units arearranged on, or substantially on the optical axis of a photoluminescentimaging system, preferably in an arrangement where the illuminationlight is directed directly towards a detector unit of an imaging system,and FIG. 7-B illustrates an arrangement where the units are arrangedsubstantially off the optical axis of a photoluminescent imaging system,preferably in an arrangement that includes a dichroic mirror reflectingthe illumination light onto the sample along the axis of an imagingsystem;

FIG. 8-A illustrates images of Jurkat, HEK293, S2 and Sf9 cell cultures,composed by the apparatus (2× magnification). Each panel shows image ofthe same cells: left; AO, right; DAPI; and FIG. 8-B illustrates imagesof Jurkat cell cultures, composed by the apparatus (2× magnification).Each panel shows image of the same cells: upper; AO, middle; DAPI,lower; superimposition of the two images;

FIG. 9 illustrates fluorescent microscopy of Jurkat cells stained withDAPI and micrographed at 40× magnification using a UV band pass filtercube. Each panel shows the following images of the same cells: left;phase contrast, right; DAPI;

FIG. 10 illustrates fluorescent microscopy of Jurkat cells micrographedat 20× magnification. A) phase contrast. B) PI stained cells (greenlongpass filter cube) C) DAPI stained cells (UV bandpass filter cube);

FIG. 11 illustrates images of isolated primary murine splenocytes,murine bone marrow cells and human blood cells, composed by theapparatus (2× magnification). Each panel shows the images of the samecells: left; AO, right; DAPI;

FIG. 12 illustrates in A) HEK293 cells stained with DACM are shown. B)HEK293 cells transfected with a CMV-RFP fusion are shown. C) Combinationof image A and B, Colour code; Blue: DACM; Red: RFP;

FIG. 13 illustrates in A) MCF-7 EC3 cells stained with PI B) MCF-7 EC3cells stained with DACM C) MCF-7 EC3 cells expressing GFP D) FIGS. A, Band C combined (colour code; red PI, Blue DACM, green GFP);

FIG. 14 illustrates a mixture of GFP transfected and untransfected MCF-7cells. Colour code; red; PI (green longpass), green; GFP (blue bandpass)and blue; DACM (UV bandpass);

FIG. 15 illustrates quantification of cellular DNA using the claimedapparatus and FACSCalibur™ flow cytometer (BD Biosciences). MCF-7 cellswere fixed with ethanol, treated with Rnase A and stained with propidiumiodide prior to analysis by the claimed apparatus (A+B) and flowcytometry (C). A) Image captured by the claimed apparatus (2×magnification) B) DNA content histogram obtained from the claimedapparatus displaying fluorescence intensity as a function of cellnumber. C) DNA content histogram obtained from FACSCalibur;

FIG. 16 illustrates a comparison of DAPI and PI for quantification ofcellular DNA using the claimed apparatus. DNA content histogram obtainedfrom the claimed apparatus displaying fluorescence intensity as afunction of cell number. Upper panel: JM cells were fixed with ethanol,treated with Rnase A and stained with propidium iodide prior to analysisby the claimed apparatus. Lower panel: JM cells were fixed with ethanoland stained with DAPI prior to analysis by claimed apparatus;

FIG. 17 illustrates DNA histograms of JM cells treated with differentdrugs. Cellular DNA content was measured using, respectively, claimedapparatus (Grey histograms) and FACSCalibur™ (white histograms);

FIG. 18 illustrates DNA histograms of yeast cells (Schizosaccharomycespombe). Cellular DNA content was measured using, respectively, claimedapparatus (Grey histograms) and FACSCalibur™ (white histograms). A)Eg892 incubated at 36° C. for 4 hours in absence of nitrogen. B) Eg544incubated at 30° C. for 3 hours in absence of nitrogen. C) Eg892incubated at 36° C. for 4 hours in presence of nitrogen. D) Eg816incubated at 36° C. for 4 hours in presence of nitrogen (different scalecompared to A-C);

FIG. 19 illustrates at Left; Apoptotic cells stained with AlexaFluor488-labelled Annexin V. Right; Non-viable cells stained with PI.Pictures captured using the claimed apparatus at 2× magnification;

FIG. 20 illustrates fluorescent microscopy and phase contrast of Jurkatcells treated with Camptothecin and stained AF-488 labelled Annexin andPI. Cells are micrographed at 40× magnification;

FIG. 21 illustrates DNA histograms of CHO cells (left panel) and JMcells (right panel) treated with different drugs. CHO and JM cells weregrown in the absence or presence of, respectively, nocodazole andcampthotecin. Cells were stained with DAPI and the DNA content wasquantified using the claimed apparatus. Cells arrested in M-phase andS-phase are indicated on the figure. Cells with fragmented DNA (lessthan 2C DNA content) are marked as sub-G₁ cells;

FIG. 22 illustrates images captured using the claimed apparatus, 2×digital and 2× optical magnification. Cells stained with anAF594-annexin V conjugate (apoptotic cells, red) and co-stained withDACM (viable cells, blue) and SYTOX green (non-viable cells, green). A)Untreated jurkat cells; less than 10% AF594-annexin V conjugate positivecells. B) Nocodazole treated jurkat cells; almost 40% AF594-annexin Vconjugate positive cells. C) and D) Result box showing the percentage ofannexin V positive cells, nonviable cell count and total cell count ofuntreated and nocodazole treated cells, respectively;

FIG. 23 illustrates images captured using the claimed apparatus, 8×magnification. Cells were stained with DACM (viable cells, blue),SR-VAD-FMK poly-caspase FLICA (apoptotic cells, red) and SYTOX green(nonviable cells green). Left column shows images of untreated CHOcells, right column shows nocodazole treated cells;

FIG. 24 illustrates Jurkat cells stained with JC-1 and DAPI. Cells weregrown in the absence (upper left) or in the presence of 10 μMcampthotecin (CPT) for 5 hours at 37° C. (upper right). Cells werestained with 2.5 μg/ml JC-1 for 10 minutes at 37° C., washed with PBS,resuspended in PBS+1 μg/ml DAPI and analysed using the claimedapparatus. In this example untreated Jurkat cells are 20%depolarized/apoptotic whereas CPT-treated Jurkat cells are 60%depolarized/apoptotic (A). Counting the number of DAPI positive cellsrevealed that the viability of the two samples is similar, beingapproximately 96% (B). (C) shows the image acquired from the CPT-treatedsample; and

FIG. 25 illustrates A) Contrast image of CD3 positive T cells from humanblood was marked using a R-phycoerythrin (PE) secondary conjugate(red/pink). To visualise all viable cells, the cell sample wasco-stained using DACM (blue). B) The histogram shows the fluorescenceintensity of PE positive cells. C) The result box summarise theinformation achieved including percentage of positive cells, total cellcount, PE mean fluorescence intensity, standard deviation and mainnumber. All data was obtained using the claimed apparatus and associatedsoftware.

DETAILED DESCRIPTION OF THE INVENTION

The object and advantages of the present invention will become apparentto those skilled in the art upon reference to the detailed descriptiontaken in conjunction with the provided figures.

According to an embodiment of the invention, the apparatus to analyse asample comprises of a sample plane onto which the sample is arranged, anexcitation light unit including at least a light source to illuminatethe sample, and a detection unit comprising at least a detector havingat least 100,000 active detection elements to detect a fluorescencesignal from the sample.

The sample to be analyzed is a solid, or substantially solid, or liquidsample. The sample contains a particle, which is selected from an animalcell, such as a mammalian, insect and fish cell, from a fungi cell, suchas a yeast cell and from a bacterium.

In an embodiment of the invention, the particle itself or materialcontained on or within the particle is photoluminescent active,generating the fluorescence signal when the sample is illuminated withthe excitation light. In another embodiment of the invention, theparticle is labelled with a photoluminescent active material, preferablya fluorescent material.

The sample or a part of sample is illuminated with the excitation lightfrom a light source, which emits light usually in a substantially narrowwavelength band. In a preferred embodiment, the light source includes alight emitting diodes and/or laser diodes for the illumination of thesample. The light source may also be selected from a solid state lightsource and a thermal light source. One highly preferred property of thesolid state light sources is its considerable illumination efficiency,particularly when illumination in a defined light band is employed. Thisis typical in fluorescence analysis, where illumination of 90% or moreof the light is preferably emitted in a waveband of less than 50 nm,such as 40 nm or less, more preferably in waveband of 20 nm of less suchas 10 nm or less, or even 5 nm or less. In particular, laser or laserdiodes wavebands of less than 5 nm are preferred, such as waveband of 2nm or lesser, preferably a waveband of 1 nm or lesser is preferred.Other highly preferred property of solid state light source is its longoperation time, e.g. effective operative life time. For example,operation time of light emitting diodes and diode lasers is longer than2,000 hours, such as 4,000 hours or more, preferably 8,000 hours ormore.

The light source is selected from a dispersive light source, an emittingdiode, a laser diode, a laser, a thermal light source, and a solid statelight source. Further, in yet another embodiment of the invention, theexcitation unit includes a plurality of light sources comprising such asat least two different light sources, such as four different lightsources, such as at least six different light sources, such as at leasteight different light sources, such as at least 10 different lightsources, emitting light of different wavelengths.

In one embodiment of the invention, the sample or a part of the samplebeing analysed is illuminated in a manner, such that the variation ofillumination intensity is lesser than a predetermined value but at thesame time, and the efficiency of illumination, defined as the fractionof emitted light from the light source illuminating a sample or sampleportion being analysed, is higher than a predetermined value.

According to an embodiment of the invention, the sample is illuminatedusing a lens unit, having a micro lens array where the micro lens arraycomprises a plurality of lenses arranged in a two dimension arrangement,to receive the excitation light and generate an illumination light thatis directed towards an illumination region of the sample plane. The lensunit produces a homogeneous illumination light to be projected on theillumination region of the sample plane with high illuminationefficiency.

In an embodiment of the invention, the excitation light focused onto themicro lens array has a divergence angle of 0.1 mrad or less, preferably0.05 mrad or less, more preferably 0.02 mrad or less. Each of the lensesin the plurality of lenses of the micro lens array produces an imagesimilar in dimensions to the region of the sample being analysed,preferably where the image produced is substantially rectangular. Theplurality of lenses of the micro lens array comprises of at least 4lenses, preferably more than 4 lenses, more preferably more than 50lenses elements, and even more preferably more than 100 lenses. Theselenses are semi-spherical lenses arranged in an array, preferably wheretwo said arrays are arranged in a series, thus forming an array oflenses. The dimension of the lens is in the range from 0.5 to 3 mm,preferably in the range from 1 to 2 mm. The size of the micro lens arrayis less than 20 mm, the size being the diameter of the lens array in thedirection perpendicular to an excitation-sample axis.

In another embodiment of the invention, the lens unit comprises of afirst micro lens array in opposite orientation with a second micro lensarray. The selection of the first micro lens array and the second microlens array is based on enhancing illumination efficiency and/or reducingillumination variation and/or integrating an optical feature of aseparate optical element into the optical effect of the lens array. Themicro lens array is housed in a casing comprising means for arrangingand fixing the micro lens array. This casing is usually made of castpolymer. The pattern of the lenses is preferably similar to the shape ofbeam of the excitation light.

In an embodiment of the invention, the illumination region is at least 2times larger than the size of emittance area of the light source. Theillumination region under investigation is only exposed to theillumination light, thereby avoiding fading of the fluorescence signal.The sample not under investigation is substantially not exposed toillumination, preferably where the illumination results in alteration inchemical and/or physical property of the sample, such as fading offluorescent signal.

The structure of the light source, with respect to the light emittingpart of the light source is of essential importance, in order to obtainillumination with high efficiency and low variation for the illuminationof photoluminescence sample material. Therefore, several highlypreferred embodiments of the present invention use “Power LED” lightsources with homogeneous emittance element, as available in severalcommercially available semiconductor light sources (e.g. LZ1-00G105 fromLedEgin, XLamp 7090 LEDs from Cree, Luxeon K2 from Lumiled, NS6B083Tfrom Nichia or the Golden Dragon (LD W51 M) from Osram). In general,several preferred embodiments of the present invention include a lightselected from a dispersive light source, an emitting diode, a laserdiode and a laser, preferably where the emittance element of substantialphysical size, such as for instance greater than 0.5 mm², or evengreater than 1.0 mm².

It is found, that when using light sources where the light emittingelements have distinct structures, such as lamp or a light emittingdiode (i.e. LED), or in general light sources, which form an image orstructure when focused, that it is possible to illuminate considerablylarge sample material, while maintaining both highly homogeneousillumination and good efficiency of emitted light. The homogenousillumination is defined by variation in illumination energy across thesample material. The illumination efficiency is defined by the ratio ofillumination light to emitted light. The present invention offerssubstantial improvement of several applications of photoluminecence,such as fluorescent microscopy. By generating strong homogeneousillumination of the sample material it is possible to perform highquality photoluminecence analysis in a faster and simple manner, thusallowing the use of more simple and robust instrumental design.

A dispersive light source, such as LED, emits light in virtually alldirections. In order to use such a light source in an application suchas fluorescence microscope, it is therefore necessary to focus the lightonto the sample material. Such focusing produces an image of the lightsource, which may be of dimensions comparable to the field of view ofthe microscope. On the other hand, the intensity of emitted light in thefield of view is an image of the light source, and therefore even thoughit is possible to illuminate the sample material with good efficiencythe intensity of the illumination varies considerably from one part ofthe sample material to another.

An often preferred embodiment of the present invention uses a number oflenses to focus light from a dispersive light source. It is oftenpreferred that such lenses is a number of substantial identical lenses,preferably lenses arranged in a lens array, such as micro lens arrays.Several preferred embodiments of the present invention use twosubstantially identical micro lens arrays, arranged in a pair withopposite orientation, while other equally preferred embodiments use asingle element, comprising micro lens arrays on two opposite surfaces.In an embodiment using a single element of micro lens arrays, the singleelement is preferably produced by a moulding method, by moulding asubstantially optically transparent material, such as glass or polymer.

A preferred property of light transmitted through a micro lens array, ishigh degree of parallel light, and therefore several preferredembodiments of the present invention include one or more lens element,such as the micro lens array, which increases the degree of parallelismof the light leaving the light source and entering the micro lens array.

In several preferred embodiments of the present invention, using microlens array elements, the micro lens arrays on both sides issubstantially identical in form, and preferably also in position, whilein other equally preferred embodiments the form and/or position of theelements of the micro lens arrays are substantially different,preferable where such difference enhances illumination efficiency and/orreduces illumination variation and/or integrates an optical feature of aseparate optical element, such as a lens, into the optical effect of themicro lens array element.

In several preferred embodiments the image of the light source producedby a lens substantially resembles the region of the sample materialwhich is under investigation, preferably by closely resembling theheight/with ratio of the detected photoluminescent region or image.

When using a number of identical lenses, it is often preferred that suchlenses are arranged in a single element, e.g. a micro lens array. Inaddition to using a micro lens array several preferred embodiments ofthe present invention further include one or more lenses, preferablywhere the purpose of such one or more lens is to collimate light fromthe light source and/or to increase the spatial angle through whichlight from the light source is collected. In other embodiment of theinvention, a collimating unit to receive the excitation light andgenerate a collimated excitation light is provided. The collimating unitincludes a lens or an array of lenses. The collimating unit increasesspatial angle through which the excitation light from the excitationunit is collected.

One often preferred property of using a number of identical lenses orlens elements, such as micro lens array, is that it is possible toeliminate the imaging of any structure in the light source, such as afilament of a lamp. If such structure is imaged on the sample materialthe intensity of illuminated light varies in accordance with the imagedstructure of the light source. Such structure can not be eliminated byconventional imaging optics and typically would require methods such asdefocusing or diffusing of light, where the result of such methods wouldgenerally result in reduction of illumination efficiency. Severalpreferred embodiments of the present invention include micro lens arrayswhich are comprised in a single element, preferably an element producedby casting optically transparent material, preferably a polymer materialin a mould.

When producing a micro lens array element by casting polymer material,it is often preferred to use considerably small thickness. Such smallthickness is typically obtained by using small lens elements, such aslens elements of 3 mm or less in diameter, such as 2 mm or less indiameter, even 1 mm or less, such as 0.5 mm or less. Typically suchmicro lens array element are less than 10 mm in thickness, such as 8 mmor less, such as 5 mm or less, even 3 mm or less. Depending on themethod used to produce the mould used for casting, it is often ofinterest to use lenses of certain diameter, such as lenses of 0.5 mmdiameter or more, such as 1 mm or more, such as 2 mm or more, such as 3mm or more. Thus it is often preferred that the diameter of the lensesis in the range from 0.5 to 3 mm, preferably in the range 1 to 2 mm.

In several preferred embodiments of the present invention, the size ofthe micro lens array is less than 20 mm, such as 15 mm or less, such as10 mm, in certain embodiments even smaller, such as 8 mm or less, suchas 5 mm or less, the size being the diameter of the lens array in thedirection perpendicular to the major axis of illumination.

Several preferred embodiments of the present invention use a singlelight source, e.g. emitting diode, laser diode or laser as a lightsource, while other equally preferred embodiments use two or more lightsources, preferably is a single assembly. In many of these embodimentsit is preferred that the two or more light sources are identical, withrespect to optical property, while in other equally preferredembodiments at least two light sources are different, with respect tooptical property. Generally two or more identical light sources are ableto generate a greater flux of light, while two or more different lightsources add flexibility with respect to properties such as wavelength ofemitted light.

In other embodiments of the invention, the two or more light sources arearranged relative to at least one other unit of the apparatus and/or tothe sample plane. Such arrangement comprises of relative movement of thelight source to at least one other unit and/or the sample plane by 1 mmor less. The arrangement arrangement affects homogeneity of theillumination light over the illumination region, preferably where anideal arrangement results in the minimum illumination variation.

Several preferred embodiments of the present invention include methodsfor production, which may place the light source relative to at leastone of the optical component, in such a manner that satisfactoryillumination efficiency and/or variation in illumination intensity isobtained.

This is preferably obtained by including means which allow the lightsource to be placed in a plane (light source plane) parallel to theillumination region plane, with accuracy in position of better than 1mm, such as better than 0.5 mm, or even better than 0.2 mm, such as withaccuracy variation of 0.1 mm or lower. Also several embodiments includemethods for production, which can place the light source and at leastone optical element in position relative to each other, in a directionparallel to the main direction of illuminated light. This is preferablyobtained by including means with allow the light source and/or at leastone optical element to be placed relative to each other, with accuracyin position of better than 0.1 mm, such as better than 0.5 mm, or evenbetter than 0.2 mm, such as with accuracy of 0.1 mm or better.

Often, it is preferred to include a wavelength separating unit, e.g. anoptical filter, in illumination means, preferably where the purpose isto define a wavelength region or polarity of illumination light. Thewavelength separation unit is a spectral filter means selected from aninterference filter, absorbing filter, and excitation filter.

One preferred embodiment of the present invention allows the lightsource to be placed close to the sample material being analysed. Thisallows the construction of compact optical system, since theillumination means, including light source and optical components can beshorter than 100 mm in length along the optical axis of the system,preferably shorter than 60 mm, more preferably shorter than 40 mm,preferably even shorter than 20 mm, such as shorter than 15 mm.

Several preferred embodiments of the present invention have theillumination means arranged on, or substantially on the optical axis ofa photoluminescent imaging system, preferably in arrangement where theflux of illumination light has a general direction toward the detectorsystem of the imaging system. Other equally preferred embodiments of thepresent invention have the illumination means arranged substantially offthe optical axis of an imaging system, preferably in arrangementincluding dichroic mirror reflecting the illumination light onto thesample along the axis of the imaging system.

One often preferred property of embodiments of the present invention isthat it is possible to illuminate sample material with homogeneous lightwith high efficiency without the use of diffuser or defocusingelement/arrangement. In another embodiment of the invention, theillumination efficiency and illumination variation is obtained withoutsubstantial defocusing of the units of the illumination means.

One often preferred property of embodiments of the present invention isthat regions of the sample material, not under investigation, aresubstantially not exposed to illumination. This is often a desirableproperty, preferably where the result of illumination is alteration inchemical and/or physical property of the sample mater, such as fading offluorescent signal.

The detection unit, comprising at least a first detector, collects thefluorescence signal, representing an image, from the sample. The firstdetector detects signals from the sample, thereby acquiring an image ofthe sample. The detector is an image sensing device, such as a ChargeCoupled Device (CCD) or a Complementary Metal Oxide Semiconductor (CMOS)device. The detector is preferably characterised by a preferreddimension of active area, preferred number of active detection elements,e.g. pixels, and preferred responsiveness, e.g. sensitivity to detectedlight. The apparatus preferably uses one or more lens for the generationof an image of the sample, where the arrangement of such lensesdetermines the focusing and optical magnification of the system.

In yet another embodiment of the invention, the detector includes anarray of active detection elements. These active detection elements arepreferably arranged in a two-dimensional arrangement allowing thesimultaneous acquisition of spatial information from the sample beinganalysed. The detector preferably comprises an array of detectionelements such as at least 100,000 active detection elements, such as400,000 active detection elements or more, such as 1,000,000 activedetection elements or more, preferably 2,000,000 active detectionelements or more. Such high number of the active detection elements isusually preferred when the analyses of the sample includes thedetermination of spatial information of biological particles in thesample. When the analysis of the sample includes the determination ofspatial information of biological particles in the sample, severalpreferred embodiments of the present sample include active detectionelements, which are less than 10 μm in size, the size being the longerof the width or the height of the detection elements, preferably wherethe size of the active detection elements is 5 μm or less, preferably 3μm or less.

In another embodiment of the invention, the apparatus includes a firstfocussing unit/means to focus the excitation light onto the sample. Inyet another embodiment of the invention, the apparatus includes a secondfocusing unit/means to focus the fluorescence signal onto the detectionunit. The first focussing unit and second focussing unit is typically alens or an array of lenses. In many preferred embodiments of the presentinvention, focusing is performed by moving one or more lenses of thefirst focussing unit and/or the second focussing unit. The focussingalso maintains a substantially fixed length of the apparatus, that is,the distance from a focal plan, where the sample is located, to an imageplan, where the detection unit is located. Preferably, the focusing isperformed by recording a series of images of a predetermined structure,where the point of focusing is determined by one or more properties ofsuch predetermined structure, such as dimension or intensity.Preferably, the apparatus is characterised in a manner, such that thearea of the field of view may be determined or estimated after takinginto account the changes in the focusing. In various embodiments of theinvention, the first focussing unit and the second focussing unitprovide a fixed optical magnification of 40× or less, preferably 20× orless, more preferably 10× or less.

In another embodiment of the invention, it is preferred that thephysical length of the apparatus is substantially same even underconditions that allow performance of different optical analysis, such asvariable wavelength in transmission or scatter microscopy, variableexcitation/emission wavelengths in fluorescence microscopy,magnification or focusing. An apparatus constructed in this manneroffers considerable mechanical advantages, such as simpler productionand improved mechanical robustness. This is achieved by using one ormore of preferred design methods, such as selection of opticalcomponents, e.g. with respect to thickness and/or curvature. Whenproducing the apparatus with a fixed physical length, it is typicallypreferred to allow for adjustment of focusing by moving of one opticalcomponent, such as a lens, along the direction of optical path. Thedegree of focusing is preferably determined by determining imagingproperty of one or more objects visible in the system, such asdetermining a size of an image of one or more objects.

Preferred embodiment of the present invention, include apparatus with afixed optical magnification, preferably where the optical magnificationlow, such as where it is less than 10 fold, such as 6 fold or less, suchas 4 fold or less, such as 2 fold or lesser. Several preferredembodiments of the present invention include apparatus, which produceimage at substantially no magnification, that is. one-to-one imaging.Typically low optical magnification is preferred when the particlesbeing analysed are approximately 20 μm or less in size.

In other equally preferred embodiments the optical magnification is 10fold or greater, such as 15 fold or greater, such as 20 fold or greater.Such optical magnification is typically preferred when analysing smallparticles, and/or when the signal detected from a particle is low, suchas when the number of detectable sites on a particle is considerablylow. Typically preferred optical magnification in such analysis is inthe range between 10 and 40 fold, such as between 15 and 30 fold.

In another embodiment of the invention, optical magnification of theapparatus, defined by the dimensions of motive and the image of themotive on the detector is of a predetermined magnitude. Themagnification may be altered by changing position of one or more unitsof the apparatus. Preferably, the magnification is changed by moving oneor more lens(es). Preferably, the magnification is changed withoutsubstantially changing the length of the apparatus, that is, thedistance from the focal plan, where the sample is located, to the imageplan, where the detection unit is located. The apparatus typicallyprovides a magnification of 40× or less, preferably 20× or less, morepreferably 10× or lesser.

In another embodiment of the invention, an excitation light filter isinserted in an excitation light path directed from at least one of thelight sources, to separate the excitation light into a plurality ofexcitation wavelength bands prior to illumination of the sample. Theexcitation light path is the path from the centre of excitation lightbeam to the sample plane

Similarly, in another embodiment of the invention, an emission lightfilter may be inserted in an emission light path directed to at leastone of the detectors, to separate the fluorescence signal into aplurality of emission wavelength bands prior to the detection of thefluorescence signal at the detection unit. The emission light path isthe path from the sample plane to the detection unit.

The excitation light filter and the emission light filter for separationof the excitation light and the fluorescence signal is selected from aninterference filter, absorption filter, low pass filter, high passfilter, and band pass filters. In several preferred embodiments, thefilters may be high-pass filters, that is, filter substantiallytransmitting light at high wavelength, while blocking light at lowwavelength, typically preferred as emission filters in fluorescencemicroscopy, low-pass filters, that is, substantially transmitting lightat low wavelength while blocking light at high wavelength, typicallypreferred as excitation filters in fluorescence microscopy. Severalpreferred embodiments include band-pass filters, e.g. substantiallytransmission in a wavelength band, while higher and lower wavelengthsare blocked, typically such filters are preferred in fluorescencemicroscopy, where the system under investigation includes multipleexcitation and/or emission possibilities.

In several highly preferred embodiments of the present systems,wavelength separation means, facilitate the investigation of two or moreproperties of a particle, such as identification of a particle orseparation of two or more groups of particles. Preferably suchidentification or separation by wavelength properties is performed byrecording two or more images of the same sample, while the sample andthe particle of the sample is/are substantially in same geometricalconfiguration in said two or more images, where said two or more imagesreflect different or substantially different wavelength properties.

In another embodiment of the invention, wavelength separation means suchas filters, facilitate investigation of two or more properties of aparticle, such as identification of a particle or separation of two ormore groups of particles. Preferably, such identification or separationby wavelength properties is performed by recording two or more images ofthe same sample, while the sample and the particle of the sample is/aresubstantially in same geometrical configuration in said two or moreimages, where said two or more images reflect different or substantiallydifferent wavelength properties.

The sample is analyzed at two or more combination of excitation andemission wavebands by selecting a combination of emission light filtersand excitation light filters. These combinations allow use of at leastone excitation or emission light filters in two or more combinationsrespectively.

In one preferred embodiment of the present invention, preferably whenrecording fluorescent information, the emission light filter is arrangedon opposite side of the sample being analysed, from the excitationsource.

Several embodiments of the present invention include an optical systemfor fluorescence analysis, where the excitation unit and the detectionunit are placed on either side of the sample plane, e.g. transmissionfluorescence, as opposed to an arrangement where the excitation unit andthe detection unit are on the same side of the sample plane and theexcitation light and the emission light are transmitted onto the samplethrough the use of minors and/or lenses, e.g. epifluorescence system.Such transmission fluorescence systems are preferred in severalembodiments, typically where they offer simpler mechanical and/oroptical arrangement. Although direct alignment of excitation source anddetector is often preferred, other embodiments, including substantialalignment, e.g. where divergence from line of parallel arrangement isgenerally less than 10 degrees, is also implemented.

In another embodiment of the invention, a sample that gives rise to aplurality of spectral information, such as the emittance of fluorescentlight at several wavelengths, is analyzed by recording two or moreimages under conditions where each recorded image substantially containsall wavelengths under consideration, but preferably in differentintensities. One preferred way of obtaining this to employ a spectralmodulation means in the optical path, preferably by using aninterferometer, such as a Michelson interferometer.

Several preferred embodiments of the present invention include means toseparate light into two or more wavelength components, where such meansinclude means which modulate light, such as a Michelson and/orFabry-Perrot interferometer. Several preferred embodiments also includemodulation means, such as interferometers, where modulation is generatedby changing optical path differences. It is preferred that such pathdifference is small, such as less than 800 μm, such as 400 μm or less,preferably 200 μm or less, more preferably 100 μm or lesser.

In other embodiments of the present invention, based on limitedwavelength resolution, the optical path difference of the modulationmeans is less than 100 μm, such as 80 μm or less, preferably 40 μm orlesser. In other embodiments of the present invention, based on moderatewavelength resolution, the optical path difference is in the range from20 μm to 200 μm, preferably in the range 40 μm to 100 μm. In severalpreferred embodiments, the wavelength resolution obtainable usingmodulation means is no more than 10 nm, such as 20 nm or smaller, suchas 40 nm, or even as small as 80 nm, the resolution being defined as thesmallest wavelength difference which can be adequately separated.

Preferred modulation means to separate light allows acquisition ofimages of modulated light at virtually any optical path difference,preferably with accuracy and resolution in position better than 1 μm,when expressed as optical path difference of the modulation means, suchas 0.5 μm or better, preferably 0.1 μm or better, more preferably 0.02μm or better. At any given optical path difference, the modulation meanscan substantially maintain optical path difference for a considerabletime, preferably for a time which is comparable to exposure time of thedetector, such as substantially maintaining optical path difference for1 ms or more, such as for 10 ms or more, preferably 50 ms or more, morepreferably 100 ms or more.

In embodiments where intensity of detected light is low, it is preferredthat modulation mean may substantially maintain optical path differenceof 200 ms or more, such as 400 ms or more, preferably 600 ms or more,more preferably 800 ms or more. When modulation means include means forthe movement of one or more optical component(s), such as a mirror or abeamsplitter, the movement is preferably brought about by one or morepiezoelectric actuator(s).

When determining wavelength properties of light using the modulationmeans, the number of images recorded at different modulation is equal tothe number of different wavelength bands of interest, preferably thenumber of images is greater than the number of different wavebands, suchas 2 times the number of wavebands, preferably 3 or 4 times the numberof wavebands. In other embodiments of the invention, the number ofimages recorded at different modulation is more than 4 times the numberof wavebands of interest, such as 8 times or more, preferably 10 timesor more.

In yet another embodiment of the invention, an actuator to move thesample plane and/or at least one of the units to modulate the lightemitted from the sample is used.

In another embodiment of the invention, two or more images of the samesample are recorded, showing different spectral properties, such asemission of fluorescence, where the position of the objects on therecorded image is substantially equal in at least two of the images.When recording images at different spectral wavelengths, such as whenrecording fluorescent intensities, this makes it possible to relatedifferent spectral information to a specific object or a particle, bycombining information from two or more images, representing different,or substantially different spectral wavelengths. When using opticalfilters to separate spectral wavelengths this feature is difficult toobtain, unless great care is given to the position and alignment of suchoptical filters, since difference in refractive index usually causesaberration and preferred embodiments of the present invention useoptical filter selection means which operate with high accuracy,preferably obtained by the use of mechanical means with smalltolerances. A preferred method of compensating for spectral aberrationis to change the position of one or more optical components, such as alens or the detectors, or the sample, in a predetermined manner, thusreducing or compensating for changes in position when recoding imagesrepresenting two or more spectral properties.

In another embodiment of the invention, an image alignment unit is used.The image alignment unit generates an aligned image from a plurality ofimages, showing different spectral information, acquired from aplurality of fluorescence signals of the sample obtained under differentor substantially different emission conditions. In another embodiment,the apparatus further comprises at least two different emission filterscapable of filtering signals emitted from the particles toward thedetector. In various embodiments of the invention, the apparatusincludes at least 4 different emission filters, such as at least 6different emission filters, such as at least 10 different emissionfilters, such as at least 15 different emission filters, such as atleast 20 different emission filters.

Where two or more emission or transmission wavebands are used for theanalysis of the same sample or sample material, it is preferred toinclude a method of image alignment. The image alignment generatesaligned images of the same sample or sample material acquired under twodifferent or substantially different emission conditions, wheredifference in the position of an object in the aligned images is lessthan 10 pixels, such as 8 pixels or less, such as 4 pixels or less,preferably even 2 pixels or less, determined as average difference inposition objects. The image alignment may be achieved by applying apredetermined transformation to a collected image, transformingcollected image to aligned image. Preferably, said predeterminedtransformation is derived from the measurement of an image comprisingobjects, which produce identifiable structure in the collected image,where said objects are visible in images collected under said differentemission conditions and maintain their position or relative positionduring the acquisition of two ore more images representing saiddifferent emission conditions. When the misalignment is relative simple,e.g. predominantly shift in position, it is preferred to use variablesrelating to each of two coordinates of the image, e.g. shift variables.However, if the misalignment includes changes in magnifications, it isoften preferred to use separate variables relating to each of the twocoordinates, e.g. factor for magnification in one direction and anotherfactor for magnification in another direction. Other equally preferredembodiments further include variables relating to both directions, e.g.variables reflecting shift and/or magnification which is determined onthe position in the image. Typically, the transformation issubstantially a shifting transformation, preferably where thetransformation can be expressed as shift of a defined number of pixelsin horizontal and/or vertical direction of the image. The degree ofshifting in the shifting transformation is substantially dependent onthe position in the image, preferably where horizontal and/or verticalshift is defined as a constant shift and variable shift determined as afraction of the index of pixel of the image. The degree of shifting inhorizontal and/or vertical direction is expressed as a polynomialfunction of the index of pixel of the image.

In yet another embodiment of the invention, a controlling unit to movethe sample plane/sample holder to a predetermined position relative tofield of view of the detection unit is used.

In one embodiment of the invention, the excitation light unit and thedetection unit are placed on either side of the sample plane. However,in yet another embodiment of the invention, the excitation light unitand the detection unit are placed on same side of the sample plane.

In another embodiment of the invention, a processor is coupled to thedetection unit to receive signal data of the fluorescence signal fromthe detection unit, to process the signal data, correlate the signaldata to a parameter to be assessed, and assess the parameter. Theapparatus further includes a means for controlling acquisition of atleast two images of the same volume of the sample, wherein informationfrom said two images represents different spectral wavelenghts.

In yet another embodiment of the invention, compensation for variablethickness of a sample holder containing a liquid sample is provided,preferably when the thickness of the sample has influence on the resultof the analysis being performed, such as the determination of the numberof particles per volume of sample. This is done by applying an insigniato the sample holder, or a part attached to the sample holder, or by inany form to correlate information concerning the physical dimensions ofthe sample holder to each measurement taken from the sample in thesample holder. In particular, when measuring at two or more separate, orpartially separate positions in the sample holder, it is preferred thatthe differences in the thickness at the separate positions may be takeninto consideration.

When determining the number of particles per volume of sample, it isnecessary to determine the volume of sample analysed. Therefore, severalembodiments of the present invention include means to registerinformation and/or determining properties concerning at least onedimension of the sample holder, preferably the thickness of the samplein the sample holder in the direction facing the detector. Theregistration of information concerning thickness preferably involvesreading an insignia reflecting a predetermination of the dimensionalproperty, e.g. the thickness. Preferably said insignia can be read byone or more sensor of the apparatus, typically where said insignia is apattern representation of information, such as binary code or barcodeinformation. Several embodiments of the present invention allow theanalysis of two or more segments of the sample holder, preferably whenthe purpose is to increase the total volume of analysed and/or to obtaina better representation of the sample being analysed, then preferablythe insignia reflects physical dimension of said two or more segments ofthe sample holder. Further embodiments of the present invention includemeans for determining physical dimension of the sample holder,preferably where it is possible to perform said determination at one ormore predetermined position(s) of the sample holder. Several preferredembodiments of the present invention read insignia and/or determinephysical dimension using a first insignia detector, preferably the firstinsignia detector is same as the detector that is used for detectingimages of the sample for the purpose of particle analysis. Preferablythe at least one registered or determined parameter has a minimumgraduation, and further where the steps of the graduation are 10% orless, when expressed as a fraction of a typical value of said parameter,preferably 8% or lesser, more preferably 6% or lesser, more preferably4% or lesser, more preferably 2% or lesser. In several preferredembodiments said graduation is in equal steps, while in other equallypreferred embodiments the graduation is in substantially unequal steps,such as when a step size is substantially equal in relative size,relative to said parameter.

In another embodiment of the present invention, a means capable ofdetermining the type of sample holder that contains the sample beinganalysed is provided. Preferably, said determination is performed byreading an insignia identifying type of said sample holder and/or bydetecting shape, size or other physical property of said sample holder.Preferably where said detection is preformed using a second insigniadetector, preferably said second insignia detector is same as thedetector that is used for detecting images of the sample for the purposeof particle analysis.

In another embodiment of the invention, a sample holder, being adaptedto hold two or more different types of sample devices, without little orno adaptation, is provided. One of said sample devices is a traditionalmicroscopy slide, or a haemocytometer chamber, such as a Büchnerchamber. Preferably, the different sample devices are placed on or in asample device holder, preferably where the sample device holder ismovable, and the movement of said sample device holder is controlled bycontrolling means, which is capable of directing the sample deviceholder in accordance with the type of sample device used.

In another embodiment of the invention, the sample holder is controlledby a controlling unit, where the detection unit is used to monitor orsense one or more information concerning the sample being analysedand/or sample device used. Preferably, the information being sensed isinformation identifying the particles in the sample or properties of theparticles in the sample and/or the sample holder or the properties ofthe sample holder. Preferably the controlling unit moves the sampleholder to one or more predetermined position(s) relative to the field ofview of the detection unit, thereby allowing the sensing of one or moreinformation. In preferred embodiments of the present invention,information being sensed is stationary information, that is, informationwhich does not change during the process of analysis, for example, thereading of an insignia, which represents a predetermined dimensionalproperty of the sample holder. While, in other embodiments, theinformation being sensed is dynamic information, for example, movementof the sample or a part of the sample device, or indication of achemical and/or physical property of the sample.

In another embodiment of the invention, resolution of the fluorescencesignal detected by the detection unit is enhanced, where resolution ofthe detected fluorescence signal is the number of significantlydifferent intervals with which the detected signal intensity isrepresented. The present invention allows the detection of signal withresolution which is greater than the nominal resolution of the detectionunit, including electrical detecting means, by recording two or moreimages of the sample where substantially only differences represented inthe two or more images is signal sensitivity, preferably obtained byvarying exposure time and/or electrical amplification of collectedsignal, and subsequently combining the two or more images to a one ormore image in a manner which represents the image of the sample with agreater image resolution than each of the two or more collected images.Therefore, the resolution in the detected signals is enhanced byproviding different signal sensitivity in the at least two images.

One aspect of the present invention relates to a system for theassessment of particles, where the assessment comprises two or moreindividual tasks, where the tasks are performed in a predeterminedmanner and where the predetermined manner comprises a number ofinstructions. Preferably the instructions can be combined in a flexiblemanner allowing the system to be adapted to perform two or moredifferent types of assessment of particles, where difference in typepreferably reflect difference in property such as timing, excitation oremission wavelength selection.

One aspect of the present invention relates to the apparatus comprisingmethods for storing collected images together with identification of thesample being analysed and method of operation of the image recordingsystem. The stored images may be arranged in a database, which allowsdata retrieval for the generation of reports and/or selection of one ormore collected images on the bases of one or more properties.

In yet another embodiment of the invention, the sample device, which maybe a vial cassette, of the described apparatus facilitates thedetermination of viability of cell suspensions from a wide range ofcultured and primary cells, by measuring cell counts (total andnon-viable) per volume.

In order to determine the viability of cells, sample of cell suspensionis drawn into the sample device by pressing the piston. The inside ofthe sample device is coated with the fluorescent dyes Acridine Orangeand 4,6-diamino-2-phenylindole (DAPI)/substituted variance of DAPI to afinal concentration of 2.5 μg/mL for both AO and DAPI/substitutedvariance of DAPI. AO stains the entire population of cells andDAPI/substituted variance of DAPI the non-viable cells. The sampledevice is placed in the apparatus where the cell counts and viabilityare determined.

The combination of acridine orange and DAPI is suitable fordetermination of the viability of cells and tissues as the two DNA dyesdoes not show spectral overlap under any circumstances. In one method ofanalysis, the cells in a suspension are incubated with the twofluorescent dyes. Acridine orange permeates all cells in the population,whereas DAPI only penetrates cells with damaged membranes. Cells labeledwith DAPI are detected by UV excitation and measuring blue light,whereas acridine orange labeled cells are detected by blue lightexcitation and measuring the emitted green light. In this manner anabsolute count of cells and percent viability can be obtained from thedata. Additionally, detection of the red light emitted by acridineorange may give further information about the cell status, vitality andintegrity.

In other embodiments of the invention, other stains of Acridine such as3,6-diamino acridine hydrochloride, 6,9-diamino-2-ethoxy-acridinelactate, quinacrine dihydrochloride, bis-N-methylacridinium nitrate,10-dodecylacridine orange bromide, quinacrine mustard dihydrochloride,acriflavine hydrochloride, and 9-isothiocyanato-10-methyl acridiniumtriflate, and 4,6-diamion-2-phenylindole (DAPI) dye/substituted varianceof DAPI to a combined concentration of 2.5 μg/mL may also be used.

In yet another aspect of the invention, a method for detecting viabilityof a test cell is provided using the apparatus of above description. Themethod comprising mixing the test cell with 4,6-diamion-2-phenylindole(DAPI) dye/or substituted variance of DAPI; allowing staining of cellsfor a time period of at the most 60 minutes, such as 30 minutes, such as20 minutes, such as at most 10 minutes; obtaining a stained cell sample,wherein only non-viable cell is stained; exposing the stained test cellto an excitation light; detecting a fluorescence signal from the stainedtest cell; and processing the fluorescence signal to identify whetherthe test cell is non-viable.

In another embodiment of the invention, the test cell may also include aplurality of cells. The cell count and non-viability of the plurality ofcells are identified by using 4,6-diamion-2-phenylindole (DAPI) dye/orsubstituted variance of DAPI. The test cell is selectedfrom a culturedcell and primary cell. The test cell is selected from a mammalian cellline, mammalian cell line in suspension, insect cell line, such asDrosophila melanogaster Schneider-2, human embryonic cell line, primarycell and human blood cell. The test cell is prepared under a controlledenvironment, as exemplified in Material and Method section in theexamples.

In yet another embodiment of the invention, Acridine Orange dye is mixedwith 4,6-diamion-2-phenylindole (DAPI) dye/substituted variance of DAPIto a combined concentration of 2.5 μg/mL. A sample device in which thetest sample is injected/loaded is coated with a combination of the AOdye and DAPI dye/substituted variance of DAPI. The Acridine Orange dyestains both viable test cell and non-viable test cell.

In yet another embodiment of the invention, the test cells areinjected/loaded into the sample device using a piston.

The excitation light is from a source selected from a thermal lightsource, such as a halogen lamp, or a gas lamp such as a xenon lamp, alight emitting diode, a laser or a laser diode or any other light sourceas described in the apparatus above.

The fluorescence signal is processed by analyzing the fluorescencesignal for number of counts of non-viable cells in the cells.

This method is performed using an apparatus that labels a test cell. Theapparatus comprises of a sample device coated at least partly with4,6-diamion-2-phenylindole (DAPI) dye/substituted variance of DAPI,wherein the DAPI/substituted variance of DAPI only stains a non-viablecell; a test cell injecting unit to load the test cell into the sampledevice; an excitation unit to expose the injected test cell to anexcitation light; a detection unit to detect a fluorescence signalemitted from the loaded test cell; and a processor to process thefluorescence signal to assess whether the test is non-viable.

In yet another embodiment of the invention, a method for providinginformation about a cell is provided using the above describedillumination apparatus and detection apparatus. The method comprises oftransfecting the cell with a fluorescent protein or fluorescentlylabelled nucleic acid; adding a thiol-reacting molecule to thetransfected cell to obtain a cell solution; exposing the cell solutionto an excitation light; detecting an emission light from the cellsolution; and processing the emission light to obtain the informationabout the cell.

The method presented here uses fluorescent proteins or fluorescentlylabeled nucleic acid in combination with a thiol-reacting molecule, suchas maleimides (e.g. DACM) and a cell-impermable DNA stain (e.g.propidium iodide) for monitoring transfection efficiency and at the sameproviding information about viability of the cell population andcytotoxity levels.

The information usually includes transfection efficiency, cell viabilityand cytotoxicity level, i.e. detecting non-viable cells.

The cell is selected from a cultured cell and primary cell. The cell isselected from a from a mammalian cell line, mammalian cell line insuspension, insect cell line, such as Drosophila melanogasterSchneider-2, human embryonic cell line, primary cell and human bloodcell.

The method further comprises of mixing a cell-impermable DNA stain. Thecell-impermeable DNA stain may be propidium iodide, and acridinehomodimer. The nucleic acid is selected from a fluorescent protein and asmall interference RNA. The thiol reacting molecule is a maleimide, suchas DACM.

In another embodiment of the invention, the cell includes a plurality ofcells. Also, the information about plurality of cells is identified byusing a combination of a fluorescent protein or fluorescently labellednucleic acid and a thiol-reacting molecule.

The excitation light is from a source selected from a thermal lightsource, such as a halogen lamp, or a gas lamp such as a xenon lamp, alight emitting diode, a laser or a laser diode or any other light sourcedescribed in the previous sections.

In several embodiments of the present invention, the excitation light isa green light and the emission light is a red light for determining anon-viable cell, when propidium iodide is used as a cell impermeable DNAstain; and the excitation light is a blue light and the emission lightis a green light for determining a non-viable cell, when acridinehomodimer is used as a cell impermeable DNA stain.

In several embodiments of the present invention, the cell expressing BFPemits a blue light when it is excited with a UV or violet light; thecell expressing CFP emits a cyan light when it is excited with a violetor blue light; the cell expressing GFP emits a green light when it isexcited with a blue light; the cell expressing YFP emits a yellow lightwhen it is excited with a blue or green light; the cell expressing dsRedor variants of this emits a red light when it is excited with a greenlight; the cell harbouring siRNA emits a blue light when it is excitedwith a UV or violet light; the cell harbouring siRNA emits a cyan lightwhen it is excited with a violet or blue light; the cell harbouringsiRNA emits a green light when it is excited with a blue light; the cellharbouring siRNA emits a yellow light when it is excited with a blue orgreen light; and the cell harbouring siRNA emits a red light when it isexcited with a green light.

The detecting comprises receiving the emission light at atleast onedetector and the processing comprises of analyzing the emission lightfor determining transfection efficiency, cell viability and cytotoxicitylevel.

The above method is implemented using an apparatus, which is used todetermine information about a cell. The apparatus includes atransfecting unit to transfect the cell with a fluorescent protein orfluorescently labelled nucleic acid; a mixing unit to add athiol-reacting molecule to the transfected cell to obtain a cellsolution; an excitation unit to expose the cell solution to anexcitation light; a detection unit to detect an emission light from thecell solution; and a processor to process the emission light to obtainthe information about the cell.

The apparatus and method of the present invention was used for analyzingparticles in a sample. The invention provides a simple, rapid andflexible device for analyzing cells at low magnification, hereundercharacterizing cells, detecting cells, counting cells as well asdetermining the viability, motility, metabolic activity, metabolitequantitation, cell division, proliferation, health, stress level,apoptosis, necrosis, other state of condition, or morphology of cells.Several examples of such analyses are presented below for betterunderstanding of the present invention:

Example 1 Focusing of Illumination Light Using Traditional Apparatus(Refer FIG. 1)

FIG. 1A shows an imaginary light source, showing substantial structurein its light emitting elements (101), comprising 4 square regionsemitting light evenly, separated and surrounded by passive elements(102), emitting no detectable light. The light source measures 1×1 mm,and the angular emission is from +40 to −40 deg.

FIG. 1B shows an arrangement, simulating the use of a light source (103,identical to item 101) for the illumination of a sample material. Thefigure shows a Biconvex Aspheric Lens (104), which has a focal lengthf=8.5 mm and a diameter D=12 mm (one such available from Melles GriotLAG000). Further the system comprises a filter element (105) typicallyused in fluorescence analysis for the elimination of light atwavelengths where emission is expected. Finally the system comprises aPlano Convex Lens (106), which has a focal length f=12 mm and a diameterD=12 mm (one such available from Edmund Optics part no 45084) andfinally the sample material (107) which is to be illuminated.

FIG. 1B shows a number of randomly selected rays and illustrates thepath of these rays through the system.

FIG. 1C shows a simulated image of the light source, as it would appearon the sample material. FIG. 1C shows strong similarity with the lightsource illustrated in FIG. 1A, where the emittance structure is easilyidentified. If suppression or elimination of the structure is wanted itbecomes necessary to use a diffusing means, e.g. by using a diffuser.This would affect the size of image and cause “blurring” resulting inbroderning of the illumination and thus loss of effective light and/ordecreased homogeneity of the illumination light.

Example 2 Focusing of Illumination Light by Micro Lens Array (Refer FIG.2)

FIG. 2A shows an arrangement, simulating illumination of a samplematerial, similar to the system in Example 1, with the addition of amicro lens array (206). The system contains a light source (201)identical to the one in FIG. 1A, biconvex aspheric lens (202), a filterelement (203), a plano convex lens (204) and finally the sample materialto be illuminated (205).

The micro lens array (206) is 12 mm in diameter, and the circumscribedsquare (12 by 12 mm) is divided into 60×44 rectangular adjacentsymmetrical biconvex lens elements with a thickness of 1.191 mm and lensradii of 0.4 mm and −0.4 mm respectively, each individual lens elementis generating a small image of the light source and when all individualimages are superimposed on the sample a rectangular shaped with homogenyillumination is present.

The system was subjected to simulation identical to the one in Example 1and the result is illustrated in FIG. 2B, which shows the illuminationof the sample material. FIG. 2B shows that the structure of the lightsource is substantially eliminated, substantially without loss ofillumination efficiency due to broadening of the illumination. Theseproperties are highly preferred when performing photoluminecentanalysis, such as fluorescence analysis.

Example 3 Comparison of Illumination (Refer FIG. 3)

FIG. 3 shows a graph, illustrating the difference of simple illuminationand illumination according to the present invention, when using a lightsource having substantial illumination structure. The comparison isbased on the results of Examples 1 and 2 as described above.

The graph in FIG. 3 shows intensity of the illumination as found inExamples 1 and 2 and indicated in FIGS. 1C and 2B, along a horizontalline indicated by the arrows in the figures.

The illumination as found in Example 1 is represented by the line 301,while the illumination as found in Example 2 is represented by the line302. The lines 303 represent the boundaries of the illumination asdefined by magnification of the optical system.

The graph illustrates that line 302 has less spread outside theboundaries 303 than does line 301, which will give greater illuminationefficiency. Further the variation in line 302 within the boundaries 303is considerably less than the variation in line 301, both with respectto the structure in the emittance of the light source, present as asignificant drop in illumination in the centre and as significantdrop-off in intensity towards the edges of the boundaries.

Example 4 Implementation According to the Present Invention (Refer FIG.4)

FIG. 4-A shows an implementation according to the present invention. Itillustrates an optical system, comprising a light source (401), forinstance ML101J17-01 (Mitsubishi, Japan), LZ1-00xx05 or LZ1-00xx03(LedEngin, USA), specifically the selection of light source depends onthe desired wavelength region and intensity. Further it comprises acollimating lens (402), e.g. LAG000 (Melles Griot, USA), an array ofmicro lenses (403), e.g. double sided lens array element, a spectralfilter means (404), typically interference and/or absorbing filters, influorescence analysis the filter is termed excitation filter, andfinally a focusing component (405), e.g. a PCX og DCX lens witheffective focal length of between 2 and 20 mm, where the effect ofchanging the focal length affects the size of the region illuminated bythe light source. Finally the illustration shows the illumination plane(406), onto which it is desired to project a relatively homogeneouslight from the light source, e.g. illumination region, preferably withrelatively high illumination efficiency.

FIG. 4B shows the implementation according to the present invention,where simulated rays of light are drawn, demonstrating the opticaloperation of the system.

Example 5 Adaptation of Illumination Region (Refer FIG. 5)

FIG. 5 shows the illumination of illumination means according to thepresent invention. The difference between FIGS. 5A and 5B shows theeffect of reducing the focal length of the focusing element (405 in FIG.4), thus affecting the effective size of the illumination region.

Example 6 Element of Micro Lenses (Refer FIG. 6)

FIG. 6 shows a micro lens array element according to the presentinvention. The element shown in FIG. 6A is a thermal plastic item,comprising means for arranging and fixing the element (601), here on theform of a rim, and two arrays of micro lenses (602), of which the arrayon the bottom is not shown.

Each of the micro lens arrays consists of a a number of 1.2×0.9 mmlenses of 1.7 mm radius, separated by about 5 mm, which is the thicknessof the micro lens element. The pattern of the micro lenses, shown inFIG. 6B, is a 13 by 9 grid, which has been roughly shaped in a circularfashion, preferably similar to the shape of the beam of light, to reducephysical size, by removing 5 lenses from each corner.

Example 7 Arrangement of Optical Module (Refer FIG. 7)

A typical embodiment according to present invention includes two or moreoptical modules and FIG. 7 shows the arrangement of these modules. Themodules are illumination module (701), sample or sample compartment(702), detection module (703) and an optical mirror (704).

FIG. 7A shows a preferred embodiment comprising an illumination, sampleand detection module, where all modules are arranged on a parallel axesrelative to each other. The illumination module, typically comprising ofa light source, focusing optic and wavelength separation means (theseitems not shown in the figure) illuminates light onto the samplecompartment, where the sample it typically arranged in a perpendicularorientation relative to the main axes of light emitted from themodulation module. Typically signals from the sample compartment areemitted in all directions from but a fraction of these signals arecollected by the detection module, which in this arrangement is on, orsubstantially on, the same axes as the illumination module and thesample compartment.

FIG. 7B shows another equally preferred embodiment of the presentinvention, where signals from the sample compartment are directedtowards the detection module by an optical minor. This embodiment allowsthe detection module to be situation outside the axes formed between theillumination and sample compartment modules, such as illustrated in FIG.7B, where the detector module is in a perpendicular arrangement.

As FIG. 7 only shows the general orientation of the different modules,and not the position of different elements, it is obvious to a personskilled in the art, that when including an optical mirror to direct thelight off the optical axis, it can often be advantageous to include anoptical component of the detection module, such as one or more lens(es)between the sample compartment module and the optical mirror. Typicallythe effect of such arrangement would be to assure a more parallel natureof the light emitted from the sample compartment, detected in thedetection module, which can render more homogeneous reaction of thelight to a wavelength dependent property, such as filtration, and/or toincrease the effective aperture of the detection module, by placing acollecting optic close to the image.

Embodiments of the present invention include two or more detectionmodules, for instance by combining the arrangement shown in FIGS. 7A andB. This is typically done by including an optical minor with wavelengthdependent reflectance properties, such that reflectance/transmittanceproperties are substantially different for different wavelengths. Suchembodiments allow simultaneous detection of two or more opticalproperties of the sample in the sample compartment module.

Example 8 Cell Count and Viability of Cells in Suspension; Jurkat (JM)Cells (a T Lymphocyte Cell Line) (Refer FIGS. 8, 9 and 10)

Materials and Methods.

Jurkat (JM) cells were cultivated at 37° C. in a humidified airatmosphere with 5% CO₂ in RPMI (Invitrogen, #61870) supplemented with10% heat-inactivated fetal bovine serum (Invitrogen, #10108-165). Jurkatcells (cell density 1.4×10⁶, 98% viability was determined using theapparatus loaded into a sample device, containing the fluorescent stainsAcridine Orange (AO) and 4,6-diamino-2-phenyindole (DAPI). The sampledevice was placed in the apparatus and the cells were counted andinvestigated using the apparatus. To show that DAPI functions to staindead cells and dead cells only, propidium iodide (PI) staining of cellswas used together with DAPI. PI is membrane impermeant and thus excludedfrom viable cells. Stained cells were also investigated using an OlympusIX50 fluorescent microscope. Images were captured using a Lumenera CCDcamera and in-house developed software. DAPI fluorescence was detectedusing a U-MNUA2 (UV band pass, 330-385 nm) filter cube (Olympus) and PIfluorescence was detected using a U-MWG2 (green longpass) filter cube(Olympus).

Results.

Both AO and DAPI stain the Jurkat cells immediately, staining the entirepopulation and the non-viable cells, respectively, whereby the viabilityof the cell population can be determined. Double staining using DAPI andPI showed that they stained the same cells, thus confirming that DAPIcan be used as a stain for nonviable cells. However, it was observedthat during incubation (more than 15 minutes) DAPI also stained a partof the PI negative population. Moreover, observing the cells at phasecontrast and under a UV filter in the fluorescent microscope, detectingthe DAPI stained cells, it was clear that the cells which appeared deadat phase contrast were also found to be DAPI positive and thus dead.

Example 9 Cell Count and Viability of Adherent Cells; HEK293 Cells (aHuman Embryonic Kidney Cell Line) (Refer FIG. 8A)

Materials and Methods.

HEK293 cells were grown at 37° C. in a humidified air atmosphere with 5%CO₂ in DMEM (Invitrogen, #31966) supplemented with 10% heat-inactivatedfetal bovine serum (Invitrogen, #10108-165). At 50% confluency cellswere harvested with 0.5 mL trypsin (Invitrogen, #25300) and neutralizedwith 5 mL medium (DMEM+10% FCS). Cells were loaded into a sample device,containing the fluorescent stains Acridine Orange (AO) and4,6-diamino-2-phenyindole (DAPI), and the cassette was placed in theapparatus. The cells were counted and investigated using the apparatusand in-house developed software.

Results.

As for the Jurkat cells, the HEK293 cells were immediately stained withAO and DAPI, staining the entire population and the non-viable cells,respectively, thus determining the viability of the cell population.

Example 10 Cell Count and Viability of Insect Cell Lines; S2 and Sf9Cell Lines (Refer FIG. 8A)

Drosophila melanogaster Schneider line-2 (S2) cells were originallyderived from late embryonic stage Drosophila embryos, and the Sf9 cellline was originally derived from pupal ovarian tissue of the Fallarmyworm Spodoptera frugiperda.

Materials and Methods.

S2 and Sf9 cells were grown at 28° C. without shaking in Schneider'sDrosophila medium (Invitrogen, #21720) and Grace's insect medium(Invitrogen, #11605), respectively, supplemented with 10%heat-inactivated fetal bovine serum (Invitrogen, #10108-165). Cells wereloaded into a sample device, which was placed in the apparatus. Thecells were counted and investigated using the apparatus.

Results.

As was the case for the suspension and adherent cell lines, the insectcell lines were also stained immediately with AO and DAPI in theVial-Cassette, showing that the Vial-Cassette can also be used tomeasure viability in insect cells.

Example 11 Cell Count and Viability of Primary Cells; MurineSplenocytes, Murine Bone Marrow Cells and Human Blood Cells (Refer FIG.11)

Materials and Methods.

The spleen from a C57BU6 mouse was placed in ice-cold PBS and gentlyground using the end of a sterile syringe. The suspension wascentrifuged at 300 g for 10 minutes; the pellet was resuspended in 1 mL0.83% NH₄Cl to lyse erythrocytes and was incubated for 3 minutes on ice.The cells were then added 14 mL PBS and centrifuged at 300 g for 10minutes. The splenocytes were resuspended in RPMI (Invitrogen, #61870)supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen,#10108-165), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen,#15140-122). The cell clumps were allowed to sediment and were removedby pipetting, and the resulting single cell suspension was used foranalysis.

The bone marrow cells were harvested aseptically in the laminar flowhood. Bilateral tibia and femur were aseptically removed, freed ofsurrounding soft tissue, and placed in a petri dish with 10 mL 70%ethanol. After 2 minutes they were transferred to ice cold PBS. The bonemarrow cavity was then flushed with 5 ml cold PBS using a 5-ml syringewith a 27-gauge needle attached, and the cells were collected from eachbone. The cells were centrifuged at 300 g for 10 min, the supernatantwas discarded, and cells were washed twice. After the second wash thecell pellet was resuspended in RPMI 1640 (Invitrogen, #61870)supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen,#10108-165), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen,#15140-122).

A small sample of human venous blood was drawn from a fingertip by useof a needle. The blood sample was diluted 5 times in PBS beforeanalysis.

A sample of each of the primary cells (murine splenocytes, murine bonemarrow cells and human blood cells) was loaded into the sample deviceand analysed in the apparatus.

Results.

As was the case for the suspension, adherent and insect cell lines, theprimary cells (murine splenocytes, murine bone marrow cells and humanblood cells) were also stained immediately with AO and DAPI in theVial-Cassette, staining the entire population and the dead population ofcells, respectively. This shows that the Vial-Cassette can also be usedto measure viability in primary cells.

Example 12 Transfection Efficiency and Cell Count of Cells ExpressingRFP Using DACM (Refer FIG. 12)

HEK293 is an embryonic human kidney cell line which in this example hasbeen transfected with a RFP (mRFP1 derived from dsRed) fused to CMVpromoter; EC41. Using DACM to stain all viable cells and RFP to detecttransfected cells, the ratio of transfected viable cells to all cells(the transfection efficiency) can easily be determined.

Materials and Methods.

HEK293 cells were cultivated at 37° C. in a humidified air atmospherewith 5% CO₂ in RPMI (Invitrogen, #61870) supplemented with 10%heat-inactivated fetal bovine serum (Invitrogen, #10108-165). 190 μL ofa mixture of transfected and untransfected HEK293 EC41 cells were added10 μL DACM (N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide, WAKOPure Chemical Industries, CAS no. 55145-14-7) dissolved in DMSO (200 μgDACM pr. mL DMSO) and mixed by pipetting. Cells were loaded into aNucleoCassette (not containing propidium iodide or other stains). TheNucleoCassette was placed in the apparatus and the cells were countedand investigated using the apparatus.

Results.

The concentration of all viable cells (based on the cells stained byDACM, FIG. 5) was determined to be 2.4×10⁶ cells pr. mL. Theconcentration of cells expressing RFP was determined to be 1.7×10⁶ cellspr. mL and the transfection efficiency was thus found to be 71.5%.

Conclusion.

Thiol-reacting probes such as DACM can be used in assays for determiningthe transfection efficiency when the transfection involves fluorescentproteins.

Example 13 Transfection Efficiency, Cell Count and Viability of CellsExpressing GFP Using DA CM and PI (Refer FIG. 13 and FIG. 14)

MCF-7 is a breast cancer cell line which in this example has beentransfected with a GFP fused to the CMV promoter. Using propidium iodide(PI) for staining non-viable cells, DACM for staining all viable cellsand GFP to detect transfected cells, the transfection efficiency, cellviability and cell count can easily be determined.

Materials and Methods.

MCF-7 cells stably expressing GFP (MCF-7 EC3) were cultivated at 37° C.in a humidified air atmosphere with 5% CO₂ in RPMI (Invitrogen, #61870)supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen,#10108-165). 190 μL MCF-7 EC3 cells were added 10 μL DACM(N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide, WAKO Pure ChemicalIndustries, CAS no. 55145-14-7) dissolved in DMSO (200 μg DACM pr. mLDMSO) and mixed by pipetting. Cells were loaded into a NucleoCassettecontaining the DNA stain propidium iodide (PI). PI is membraneimpermeant and is therefore excluded from viable cells. TheNucleoCassette was placed in the apparatus and the cells were countedand investigated using the apparatus. The cells were also investigatedusing an Olympus IX50 fluorescent microscope, and images were capturedusing a Lumenera CCD camera and in-house developed software. PI, DACMand GFP fluorescence were detected using, respectively, U-MWG2 (greenlong pass: 510-550 nm), U-MNUA2 (UV band pass, 330-385 nm) and (bluelong pass) filter cubes (Olympus).

Results.

A low fraction of the cells were stained by PI (FIG. 6A) and theapparatus determined the concentration of non-viable cells to be 1.2×10⁵cells pr. mL. The concentration of viable cells (the cells stained byDACM, FIG. 6B) was determined to be 2.7×10⁶ cells pr. mL. Based on this,the viability of the cells in the sample was found to be 95.6%. Theconcentration of cells expressing GFP was determined to be 2.6×10⁶ cellspr. mL and the transfection efficiency was thus found to be 96%.

A mixture of transfected and untransfected MCF-7 cells were alsoinvestigated using a fluorescent microscope (FIG. 7). The figure consistof an overlay of images of the same cells captured using differentfilter cubes for detection of DACM (UV band pass), GFP (blue band pass)and PI (green long pass). As can be seen from the figure, DACM stainsviable cells including the cells expressing GFP, while dead cells areonly stained by PI.

Conclusion.

Thiol-reacting probes such as DACM can be used in an assay fordetermining transfection efficiency and combined with an impermeablestain such as PI, information about viability of the cells investigatedcan also be obtained.

Example 14 Quantification of DNA Content Using DAPI or Propidium Iodide(PI) in Mammalian Cells (Refer FIG. 15 and FIG. 16)

The intensity of fluorescence integrated over a DAPI stained cell is instoichiometric relationship to DNA content. DAPI preferentially binds todouble stranded DNA and the quantum yield of DAPI/RNA complexes is only20% of that of the DAPI/DNA complex. Hence, using DAPI, there is norequirement for removing RNA by Rnase treatment prior to DNA contentmeasurements. This is a prerequisite for other dyes commonly used formeasurements of cellular DNA content, such as propidium iodide. DAPIinteracts with double stranded DNA by associating with AT clusters inthe minor groove. When bound to double-stranded DNA its absorptionmaximum is at 358 nm and its emission maximum is at 461 nm. Binding ofDAPI to DNA produces a 20-fold fluorescence enhancement.

In the example, the DNA content of an adherent cell lines, MCF-7, asuspension cell line, JM (a jurkat derived cell line), was measuredusing either DAPI or propidium iodide as DNA stain.

Materials and Methods.

MCF-7 cells were grown in RPMI+10% FCS to 80-90% confluency and cellswere washed once in PBS, trypsinized and harvested by centrifugationprior to ethanol fixation. JM cells were grown in RPMI+10% FCS to adensity of 5.0×10⁵ cells/ml. Cells were harvested by centrifugation andwashed once in PBS prior to ethanol fixation. MCF-7 and JM cells werethoroughly resuspended in 0.5 ml PBS and the cell suspensions were eachadded to 4.5 ml of ice-cold 70% ethanol fixative. Cells were kept infixative for at least two hours before staining with either DAPI or PI.

For PI Staining.

Cells were harvested by centrifugation for 5 min at 200×g and washedonce with PBS. After another centrifugation cells were resuspended inpropidiun iodide staining solution (To 100 ml of 0.1% (v/v) Triton X-100in PBS add 20 mg DNase-free RNase A and 2 mg of propidium iodide) andincubated for 30 minutes at 37° C. prior to analysis by flow cytometryand the claimed apparatus.

For DAPI Staining.

Cells were harvested by centrifugation for 5 min at 200×g and washedonce with PBS. After another centrifugation cells were resuspended inDAPI staining solution (To 100 ml of 0.1% (v/v) Triton X-100 in PBS add0.1 mg 4′,6-diamidino-2-phenylindole; DAPI) and directly analysed by theclaimed apparatus.

Results.

Rnase A treated and PI stained MCF-7 cells were analysed by the claimedapparatus and standard flow cytometry. As shown in FIG. 15, the claimedapparatus provides accurate and precise data on cellular DNA contentthat is comparable with that obtained by flow cytometry, the method ofwhich is the most commonly used for quantification of DNA. Next, MCF-7cells from the same culture were stained directly with DAPI (no Rnase Atreatment) and were analysed by using the claimed apparatus. As shown inFIG. 16, the DNA content histogram obtained from, respectively, PI andDAPI stained cells are almost identical. Hence, the claimed apparatusenables quantification of cellular DNA using both PI and DAPI staining.

Example 15 Effects of Drugs on DNA Content in Mammalian Cells (ReferFIG. 17)

The DNA content of JM cells treated with different drugs was measuredusing propidium iodide as DNA stain.

Materials and Methods.

JM cells were grown in RPMI+10% FCS to a density of 4.7×10⁵ cells/ml andthen either serum starved or treated with the following drugs for 20hours:

1. Nocodazole (G₂-M arrest): 0.5 μM 2. L-mimosine (G1): 0.5 mM 3.Hydroxyurea, HU (S): 2 mM 4. Camptothecin, CPT (S): 5 μM 5. Etoposide(S): 20 μM 6. Untreated control

Cells were harvested by centrifugation and washed once in PBS prior toethanol fixation. Cells were thoroughly resuspended in 0.5 ml PBS andthe cell suspensions were each added to 4.5 ml of ice-cold 70% ethanolfixative. Cells were kept in fixative for at least two hours beforestaining with PI.

For PI Staining.

Cells were harvested by centrifugation for 5 min at 200×g and washedonce with PBS. After another centrifugation cells were resuspended inpropidiun iodide staining solution (To 100 ml of 0.1% (v/v) Triton X-100in PBS add 20 mg DNase-free RNase A and 2 mg of propidium iodide) andincubated for 30 minutes at 37° C. prior to analysis by flow cytometryand the claimed apparatus.

Results.

JM cells treated with drugs affecting different phases of the cell cyclewere analysed by the claimed apparatus and standard flow cytometry. Asshown in FIG. 17, the claimed apparatus provides accurate and precisedata on cellular DNA content that is comparable with that obtained fromflow cytometry. Hence, the claimed apparatus enables quantification ofcellular DNA and may be used for evaluating the effects of differentdrugs on the cell cycle.

Example 16 Quantification of DNA Content Using Propidium Iodide in YeastCells (Refer FIG. 18)

The DNA content of yeast cells (Schizosaccharomyces pombe) was measuredusing propidium iodide as DNA stain. The following two strains wereused:

1. Eg544 (CM-SP4), h⁻ Δmat2,3

2. Eg816 (CM-SP7), h⁻ cdc25-22 leu1-32

3. Eg892 (CM-SP27), h⁺ cdc10-V50 ura4-D18

Cdc cells (Eg816 and Eg892) were grown to a density of 5×10⁶ cells/ml inEMM medium at 25° C. and then incubated at 36° C. (restrictivetemperature of cdc mutants) for 4 hours before harvesting and fixationin 70% ethanol. Cdc10 cells were incubated at restrictive temperatureboth in the presence and absence of nitrogen (EMM-N).

Wild type cells (Eg544) were grown to a density of 5×10⁶ cells/ml in EMMmedium at 30° C., then shifted to EMM and EMM-N(nitrogen starvation) andincubated at 30° C. for 3 hours before harvesting and fixation in 70%ethanol.

Expected arrest point of various cdc mutants at restrictive temperatureand of cells starved of nitrogen:

-   -   1. Cdc10-V50=G₁ arrest: cdc10-V50 cells grown at the restrictive        temperature will arrest in G₁ with a 1C DNA content. The        cdc10-V50 allele is leaky and a minority of the cells in the        population will still be dividing. To get a complete G₁ arrest        the cdc10-V50 cells have to starved of nitrogen.    -   2. Cdc25-22=G2 arrest=Cdc25-22 cells grown at the restrictive        temperature will arrest in G₂ with a 2C DNA content.    -   3. Nitrogen starved cells=G₁ arrest    -   4. Vegetatively growing wild type cells (Eg544) spend most of        their time in the G2 phase of the cell cycle and, hence, the        majority of the cells in the population will have a 2C DNA        content. Upon nitrogen starvation wild type cells will arrest in        G₁ with a 1C DNA content. A complete G₁ requires numerous hours        of starvation. In this experiment the cells are only starved for        3 hours and therefore only few cells will be arrested in G₁ with        a 1C DNA content.

DNA Content Analysis.

About 3×10⁶ ethanol-fixed cells were Rnase-treated (0.1 mg/ml RNase A in50 mM sodium citrate, pH 7.0 over night at 37° C.), stained withpropidium iodide (4 μg/ml PI in 50 mM sodium citrate, pH 7.0), sonicatedfor 10 seconds and analysed by flow cytometry (10.000 cells were countedusing a Becton-Dickinson FACSCalibur) and the claimed apparatus.

Results.

Yeast cells arrested at different stages of the cell cycle were analysedby the claimed apparatus and standard flow cytometry. As shown in FIG.18 the claimed apparatus provides accurate and precise data on cellularDNA content that is comparable with that obtained by the flow cytometry.Hence, the claimed apparatus enables quantification of DNA content inyeast cells and can be used for studying e.g. regulation of the cellcycle.

Example 17 Apoptosis Assays Annexin V (Refer FIG. 19 and FIG. 20)

An assay for detection of early apoptosis is based on the fact thatphosphatidylserine (PS) in healthy, non-apoptotic cells predominantlyare located on the internal leaflet of the plasma membrane facing thecytosol. Early in the apoptotic process while the cell membrane arestill intact, the PS are translocated to the outer layer of themembrane. Annexins are group of cellular proteins that bind tophospholipids in a calcium-dependent manner, and a member of this group;Annexin V has proven to be a useful tool in detecting apoptotic cellssince it preferentially binds to negatively charged phospholipids likePS and shows minimal binding to phosphatidylcholine and sphingomyeline.

By conjugating a fluorescent label to Annexin V it is possible toidentify and quantitate apoptotic cells. Annexin V will also bind to PSon late apoptotic and necrotic cells but as the membrane integrity onthese cells has been lost, these can be distinguished from earlyapoptotic cells by the use of an impermeant dye such as PI or DAPI.

Here the claimed apparatus is used in assay fluorescently labeledAnnexin V to stain apoptotic and necrotic cells and PI to distinguishbetween the early and late apoptotic/necrotic cells.

Materials and Methods.

Jurkat (JM) cells were cultivated at 37° C. in a humidified airatmosphere with 5% CO₂ in RPMI (Invitrogen, Cat. No. 61870) supplementedwith 10% heat-inactivated fetal bovine serum (Invitrogen, Cat. No.10108-165). 3 μM Camptothecin (Sigma, Cat. No. C-9911) was added to thecells in order to induce apoptosis. After three hours incubation (37°C., 5% CO₂) cells were harvested in cold phosphate-buffered saline (PBS)and stained with AlexaFluor 488-labelled Annexin V from the Vybrant®Apoptosis Assay Kit #2 (Molecular probes, V13241) according to themanufacturer's protocol. The stained cells were loaded into a cassettecontaining propidium iodide. The cassette was placed in the claimedapparatus and the cells were counted and investigated using the claimedapparatus and in-house developed software. The cells were alsoinvestigated using an Olympus IX50 fluorescent microscope. Images werecaptured using a Lumenera CCD camera and in-house developed software.AF488 fluorescence was detected using a U-MWIBA3 (blue bandpass) filtercube (Olympus) and PI fluorescence was detected using a U-MWG2 (greenlongpass) filter cube (Olympus).

Results.

Using the software associated with the claimed apparatus, theconcentration of apoptotic cells (Annexin V conjugate binding cells) wasdetermined to be 2.6×10⁵ while the concentration of non-viable cells wasfound to be 4.5×10⁴ (see FIG. 19). Investigating the cells underfluorescence microscope three types of cells was found: Cells onlyvisible at phase contrast (viable cells), cells positive for both PI andfluorescently labeled Annexin V (necrotic or late apoptotic cells) andfinally cells excluding PI but positive for staining with fluorescentlylabeled Annexin V (early apoptotic cells) (see FIG. 20).

Alternative Methods.

An alternative method for determining the ratio of apoptotic cells tothe total number of cells is combining the above described method ofExample 17 with a stain for all cells such as acridine orange (AO) or astain only staining viable cells such as DACM. For example, all cellsare stained with AO or another compound staining all cells, combinedwith fluorescently labelled Annexin V to stain apoptotic cells and animpermeant stain, staining non-viable cells or all viable cells arestained with DACM or another thiol reacting probe, combined withfluorescently labelled Annexin V to stain apoptotic cells and animpermeant stain, staining non-viable cells.

Example 18 Detection Early Apoptosis by Monitoring Changes in theMitochondrial Membrane Potential

A very early event in apoptosis is collapse of the electrochemicalgradient across the mitochondrial membrane followed by uncoupling of therespiratory chain. In order to distinguish healthy and apoptotic cells asimple assay based on this phenomenon is to stain the cells usingcationic dyes such as5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1) or tetramethylrhodamine methyl ester perchlorate or othercompounds which fluoresces differently in the two populations. One ofthe examples is JC-1 in healthy, non-apoptotic cells found in themitochondrial matrix where it forms red fluorescent aggregates. However,in the event of a fall in the mitochondrial membrane potential (e.g. inconnection to apoptosis) the dye returns to its monomeric form which isassociated with a large emission shift to green.

For the analysis of changes in mitochondrial membrane potential andhence apoptosis, the cells are incubated with the chosen cationic dye(e.g. JC-1) and analyzed at relevant wave lengths using the claimedapparatus. If, for example, using JC-1 the shift from red to green arequantitated in order to determine the apoptotic cell population. A cellimpermeable DNA stain (like DAPI) may be included to stain necroticcells.

Example 19 Caspase Based Assays (Refer FIG. 23)

Another early marker of apoptosis is the activity of caspases. Caspasesare a family of cysteine aspartic acid specific proteases. The caspasesmediate cell death and play essential roles in apoptosis, necrosis andinflammation. Caspases are regulated at a post-translational level, andcan thus be rapidly activated. The recognition site for caspases ismarked by three to four amino acids followed by an aspartic acidresidue, with the cleavage occurring after the aspartate. Caspases areactivated by proteolytic cleavage of a precursor, and since the cleavagesite within precursors matches the specificity of caspases, sequentialactivation of precursors by activated caspases can occur.

A way to measure the activity of the different types of caspases is touse a peptide substrate containing the recognition site of the caspaseof interest linked to a probe which changes fluorescent properties aftercleavage at the aspartic acid residue.

A key mediator of apoptosis is caspase-3 (CPP32, apopain, YAMA) whichamplifies the signal from initiator caspases such as caspase-8. Anexample of a fluorogenic substrate for caspase-3 is acetylAsp-Glu-Val-Asp (acetyl-DEVD) 7-amido-4-methylcoumarin, which uponcleavage results in the release of the fluorescent7-amino-4-methylcoumarin (AMC) moiety. The caspase activity can thus bequantitated be measuring the fluorescence. The activity of othercaspases can also be determined applying the same concept.

Using the claimed apparatus to quantitate caspase activity by thismethod, the cells are permeabilised/lysed and incubated with thefluorogenic substrate corresponding to the caspase(s) of interest. Afterincubation fluorescence of the cells are determined using the claimedapparatus and used as a measure for caspase activity.

Another caspase based apoptosis assay is the FLICAssay (FluorochromeInhibitor of Caspases Assay). This assay detects active caspases insidethe cell using a caspase specific inhibitor sequence linked to afluorescent probe. The non-cytotoxic caspase specific inhibitor is cellpermeant and passes through the intact plasma membrane and covalentlybinds to the reactive cysteine residue on the large subunit of theactive caspase heterodimer. Unbound caspase specific inhibitor diffusesout of the cell and is washed away, thus there is no interference frompro-caspases or inactive forms of the enzyme. Measuring the fluorescencethus gives a direct measure of the amount of active caspase in the wholeliving cell.

To quantify caspase activity by the FLICAssay, the cells to beinvestigated are incubated with the chosen caspase specific inhibitorsequence linked to a fluorescent probe. Cells are washed to removeunbound inhibitor and the fluorescence of the cells is determined usingthe claimed apparatus thereby making measurement of caspase activity onthe single cell level possible. Stains for viable/all cells such as DACMor AO and/or nonviable cells such as DAPI/PI may be included in theassay.

Materials and Methods.

CHO cells were cultivated in RPMI (Invitrogen, Cat. No. 61870)supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen,Cat. No. 10108-165) at 37° C. in a humidified air atmosphere with 5%CO₂. At 80% confluency cells were either treated with 1 μM nocodazole(Sigma, Cat. No. M1404) or left untreated. After 16 hours incubationcells were harvested in cold phosphate-buffered saline (PBS) and stainedwith SR-VAD-FMK poly-caspase FLICA (Immunochemistry Technologies, #91)according to the manufacturer's protocol. After staining all viablecells were co-stained using DACM(N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide, WAKO Pure ChemicalIndustries, CAS no. 55145-14-7) and nonviable cells were stained withSYTOX green (Invitrogen, S7020). The stained cells were loaded into aNC-Slide. The NC-Slide was placed in the claimed apparatus and the cellswere counted and investigated using the claimed apparatus and in-housedeveloped software. The cells were also investigated using an OlympusIX50 fluorescent microscope. Red fluorescent (apoptosis positive) cellswere detected using a U-MWG2 (green longpass) filter cube (Olympus),DACM positive cells were detected using the U-MNUA2 (UV band pass)filter cube (Olympus) and SYTOX green fluorescent cells were detectedusing a U-MWIBA3 (blue bandpass) filter cube (Olympus).

Results.

Using the software associated with the claimed apparatus, theconcentration of apoptotic cells—based on caspase activity—wasdetermined for both the nocodazole treated and the untreated cells.Substantially more nocodazole treated cells exhibited strong redfluorescence than untreated cells (see FIG. 23). This observation wassupported by visual inspection using the fluorescence microscope

Example 20 TUNEL

A characteristic of late stage apoptosis is the fragmentation of nuclearchromatin which results in a multitude of 3′-hydroxyl termini of DNAends. Thus, a method commonly used for detection of late apoptosis isTUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick EndLabeling). The TUNEL method involves terminal deoxynucleotidyltransferase to transfer biotin-dUTP or fluorescently marked dUTP to thestrand breaks of cleaved DNA. When using biotin-dUTP the biotin-labeledcleavage sites are further reacted with fluorescently conjugated avidinor streptavidin (e.g. FITC avidin) to enable detection andquantification of DNA degradation and thus apoptosis. Usingfluorescently marked dUTP this is done directly. Non-apoptotic cellsincorporate low levels of labelled-dUTP due to absence of exposed3′-hydroxyl DNA ends in these cells.

As the terminal deoxynucleotidyl transferase has to enter the cell, theplasma membrane has to be permeabilized prior to the enzymatic reaction.To avoid that this leads to loss of the fragmented DNA, the cells arefixed with either formaldehyde or glutaraldehyde beforepermeabilization. This fixation crosslinks the DNA to other cellularcomponents and precludes its extraction during permeabilization. Thusthe procedure for detecting late apoptosis using the claimed apparatusis as follows: The cells to be investigated are fixated and thenpermeabilized. The cells are then incubated with terminaldeoxynucleotidyl transferase and dUTP labeled with the preferred marker(biotin, fluorescent probe or antibody). During the incubation theterminal deoxynucleotidyl transferase catalyzes the binding of dUTP tothe 3′-hydroxyl ends in the DNA. After washing the stained cells areinvestigated using the claimed apparatus thereby making measurement ofapoptosis on the single cell level possible. Stains for all cells suchas DAPI, PI or AO may be included in the assay (As the cells arepermeabilized permeant as well as impermeant stains may be use).

Example 21 Cell Proliferation

Cell proliferation is the measurement of the number of cells that aredividing in a culture. Rapid and accurate assessment of cellproliferation is a requisite in many experimental set-ups and is veryuseful for evaluating e.g. the cytotoxic, mutagenic and carcinogeniceffects of chemical compounds on eukaryotic cells and for estimatingcell doubling time and verify the health of cell cultures. One way ofestimating this parameter is by measuring growth curves, however, thisis both tedious and time consuming. A second way to analyze cellproliferation is by using DNA synthesis as a marker for proliferation.In these assays, the incorporation of nucleotide-analogues into DNA isquantified. Incorporation of the nucleotide-analogue into DNA isdirectly proportional to the amount of cell division occurring in theculture.

In one method of analysis cells are incubated with bromodeoxyuridine(BrdU) during the final 2 to 24 hours of incubation. BrdU will beincorporated into the DNA of dividing cells and will be detected andquantified using an anti-BrdU antibody. In order to facilitate antibodybinding to the incorporated BrdU, cells are permeabilized and the DNA isdenatured by a one-step fixative/denaturing solution. After antibodyincubation unbound antibody is washed away and a fluorochrome-conjugatedsecondary antibody (e.g. FITC labelled antibody) which recognizes theanti-BrdU antibody is added to the solution. Unbound secondary antibodyis washed away and the cellular fluorescence is detected and quantifiedusing the claimed apparatus. The intensity of the fluorescent signal isproportional to the amount of incorporated BrdU in the cells. Inaddition to evaluation of cell proliferation, information about e.g.cell number and cellular antigens can be obtained from the same sampleat the single cell level.

A third way to analyse cell proliferation involves labelling of cellswith a fluorescent dye that is retained within the cell withoutaffecting cellular function. For each round of cell division, theintensity of the fluorescence is decreased by half.

In one method of analysis cells are incubated with amine reactivediacetate succinimidyl esters (commonly referred to as SE), thatdiffuses freely into cells. The fluorescent dyes react withintracellular proteins as well as proteins present on the cell surface.Upon reaction the fluorescent dyes are covalently linked to the proteinsand are, thus, retained within the cell. The cellular fluorescence isdetected and quantified using the claimed apparatus and as a celldivides the fluorescence intensity is decreased by half. In addition toevaluation of cell proliferation, information about e.g. cell number,viability and cellular antigens can be obtained from the same sample atthe single cell level. E.g. a cell impermeable DNA stain (like DAPI) canbe included in the assay to discern the percentage of cells that aredead.

Example 22 Detection and Quantification of Flourescent Proteins,Hereunder FRET Analysis

A wide range of fluorescent protein variants have now been developedthat feature fluorescence emission spectral profiles spanning almost theentire visible light spectrum. Mutagenesis of the original greenfluorescent protein (GFP) isolated from the jelly fish Aequorea victoriahave resulted in new fluorescent probes that range in color from blue toyellow and are some of the most widely used in vivo reporter moleculesin biological research. Longer wavelength fluorescent proteins, emittingin the orange and red spectral regions, have been developed from themarine anemone Discosoma striata and reef corals belonging to the classAnthozoa. The claimed apparatus can detect the known fluorescentproteins, including BFP, CFP, GFP, GFP-uv, YFP, HcRed1, KFP1, mRFP1,mCherry and other variant derived from dsRED.

In one method of analysis the coding region of the fluorescent proteinis linked to a promoter (or enhancer) from a gene of interest. Thefusion construct is transfected into a host cell and the fluorescenceemitted by the produced fluorescent protein is readily detected andquantified by the claimed apparatus. The intensity of the fluorescentsignal is proportional to the expression level of the fluorescentprotein, which again is a measure of the activity of the gene ofinterest. Hence, this method can be used to study transcriptionalactivity of a gene of interest under various growth conditions.

In a second method of analysis the coding region of the fluorescentprotein is linked to the coding region or part of the coding region froma gene of interest. The construct is transfected into a host cell andthe fluorescence emitted by the produced fusion protein is detected andquantified by the claimed apparatus. The intensity of the fluorescentsignal is proportional to the expression level of the fluorescent fusionprotein, which again is a measure of the level of the protein ofinterest. This method can be used to study dynamic of a protein ofinterest, such as measurement of protein stability and protein half-lifeunder various growth conditions.

The use of fluorescent protein enables the study of dynamic molecularprotein-protein interactions within living cells. One way of detectingmolecular interactions involves fluorescence resonance energy transfer(FRET) between two fluorescent proteins or between a single fluorescentprotein and a second fluorophore. For example, YFP and CFP can functionas a donor-acceptor pair for FRET, in which excitation of the donor(cyan) molecule leads to emission from the acceptor (yellow) molecule,provided that the proteins are close enough for energy transfer tooccur. FRET can therefore be used to monitor direct protein-proteininteractions between EYFP and ECFP fusion proteins in cells. In onemethod of analysis the coding region of Cyan Fluorescent Protein (CFP)is linked to the coding region or part of the coding region from onegene of interest. Likewise, the coding region of Yellow FluorescentProtein (YFP) is linked to the coding region or part of the codingregion from a second gene of interest. The two fusion proteins areco-expressed in a host cell. CFP is excited by violet light and yellowlight emitted from YFP is detected and quantified.

Example 23 Detection and Quantification of DNA and RNA Markers UsingFISH

Fluorescence microscopy at low magnification is very useful forobtaining precise and accurate information about gene amplification andexpression levels of RNA markers in cell populations using fluorescentin situ hybridisation (FISH).

In one method of analysis cells are permeabilized and the DNA isdenatured by a one-step fixative/denaturing solution facilitating thehybridisation of specific nucleotide probes, such as DNA, RNA, PNA andLNA, labelled with a fluorochrome (e.g. FITC labelled antibody). Afterhybridisation unbound fluorochrome-conjugated probe is washed away andthe cellular fluorescence is detected and quantified using the claimedapparatus. The intensity of the fluorescent signal is proportional tothe amount of the DNA or RNA marker present in the cells. The method canbe used for detection and quantification of DNA amplification, e.g. HER2and TOP, and of RNA levels, e.g TERT mRNA. In addition to evaluation ofDNA and RNA markers, information about e.g. cell number, DNA content andantigens can be obtained from the same sample at the single cell level.

Example 24 Effects of Drugs on DNA Content in Mammalian Cells (ReferFIG. 21)

The DNA content of CHO cells treated with Nocodazole (inhibitor ofM-phase) and JM cells treated with camptothecin (inhibitor of S-phase)was measured using DAPI as DNA stain.

Materials and Methods.

CHO cells were grown in RPMI+10% FCS to 80-90% confluency and treatedwith 0.5 μM nocodazole for 24 hours. Cells were washed once in PBS,trypsinized and harvested by centrifugation prior to ethanol fixation.JM cells were grown in RPMI+10% FCS to a density of 5.0×10⁵ cells/ml andtreated with 5 μM camptothecin for 16 hours. Cells were harvested bycentrifugation and washed once in PBS prior to ethanol fixation. CHO andJM cells were thoroughly resuspended in 0.5 ml PBS and the cellsuspensions were each added to 4.5 ml of ice-cold 70% ethanol fixative.Cells were kept in fixative for at least two hours before staining withDAPI. For DAPI staining cells were harvested by centrifugation for 5 minat 200×g and washed once with PBS. After another centrifugation cellswere resuspended in DAPI staining solution (To 100 ml of 0.1% (v/v)Triton X-100 in PBS add 0.1 mg 4′,6-diamidino-2-phenylindole; DAPI) anddirectly analysed by the claimed apparatus.

Results.

DNA content of CHO and JM cells treated with drugs affecting differentphases of the cell cycle were analysed by the claimed apparatus. Asshown in FIG. 21, the claimed apparatus provides accurate and precisedata on cellular DNA content and can be used for evaluating the effectsof different drugs on the cell cycle. Moreover, the claimed apparatuscan be used for identifying cells with fragmented DNA (so-called sub-G₁cells). DNA fragmentation is one hallmark of apoptotic cell death.

Example 25 Apoptosis Assays Annexin V (Refer FIG. 22)

Here the claimed apparatus is used in assaying fluorescently labelledannexin V to stain apoptotic and necrotic cells and SYTOX green todistinguish between the early and late apoptotic/necrotic cells. Inaddition DACM is used to stain all viable cells.

Materials and Methods.

Jurkat cells were cultivated at 37° C. in a humidified air atmospherewith 5% CO₂ in RPMI (Invitrogen, Cat. No. 61870) supplemented with 10%heat-inactivated fetal bovine serum (Invitrogen, Cat. No. 10108-165).Cells were either treated with 1 μM nocodazole (Sigma, Cat. No. M1404)or left untreated. After 16 hours incubation (37° C., 5% CO₂) cells wereharvested in cold phosphate-buffered saline (PBS) and stained withAlexaFluor 594-labelled annexin V (Molecular probes, A-13203) accordingto the manufacturer's protocol. After annexin V staining all viablecells were co-stained using DACM(N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide, WAKO Pure ChemicalIndustries, CAS no. 55145-14-7) and nonviable cells were stained withSYTOX green (Invitrogen, S7020). The stained cells were loaded into aNC-Slide. The NC-Slide was placed in the claimed apparatus and the cellswere counted and investigated using the claimed apparatus and in-housedeveloped software. The cells were also investigated using an OlympusIX50 fluorescent microscope. SYTOX green fluorescence was detected usinga U-MWIBA3 (blue bandpass) filter cube (Olympus), the annexin V AF594conjugate was detected using a U-MWG2 (green longpass) filter cube(Olympus) and DACM was detected using the U-MNUA2 (UV band pass) filtercube (Olympus).

Results.

Using the software associated with the claimed apparatus, theconcentration of apoptotic cells (annexin V conjugate binding cells) wasdetermined for both the nocodazole treated and the untreated cells.Nonviable cells were gated out based on SYTOX green staining. Theanalysis of the viable cells showed that while less than 10% of thecontrol cells were annexin V positive more than 40% of the nocodazoletreated cells were annexin V positive (See FIG. 22), while viability wasalmost not affected. This observation was supported by visual inspectionusing the fluorescence microscope.

Example 26 Detection Early Apoptosis by Monitoring Changes in theMitochondrial Membrane Potential (Refer FIG. 24)

Loss of the mitochondrial membrane potential is known to precedeapoptosis and chemical-hypoxia-induced necrosis. The lipophilic cationicdye JC-1(5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide)display potential-dependent accumulation in the mitochondria andprovides a simple, fluorescent-based method for distinguishing betweenhealthy and apoptotic cells. In healthy cells, the negative chargeestablished by the intact mitochondrial membrane potential facilitatesthe accumulation of JC-1 in the mitochondrial matrix. At highconcentrations JC-1 forms aggregates and become red fluorescent. Inapoptotic cells the mitochondrial potential collapses and JC-1 localizesto the cytosol in its monomeric green fluorescent form. A cellimpermeable DNA stain (like DAPI) can be included to stain necrotic andlate apoptotic cells.

Materials and Methods.

Jurkat cells were grown in RPMI+10% FCS to a density of 5.0×10⁵ cells/mland treated with 10 μM campthotecin (apoptosis inducing drug) for 5hours. Cells were stained with 2.5 μg/ml JC-1 for 10 minutes at 37° C.,washed with PBS, resuspended in PBS+1 μg/ml DAPI and analysed using theclaimed apparatus.

Results.

Jurkat cells stained with JC-1 and DAPI were analysed by the claimedapparatus (FIG. 24). Red and green fluorescence were quantified toidentify apoptotic cells (FIG. 24A). To estimate the number ofnectrotic/late apoptotic cells DAPI (blue) positive cells were counted(FIG. 24B). As shown in FIG. 24, the claimed apparatus can be used toidentify cells with collapsed mitochondrial membrane potential andenables the identification of early apoptotic cells.

Example 27 Detection and Quantification of Antigens (Refer FIG. 25)

Fluorescence microscopy at low magnification is very useful forobtaining precise and accurate information about the expression level ofantigens, such as intracellular and cell surface proteins, in cellpopulations.

An example of this is described in the following. Cells are incubatedwith a primary antibody recognizing an antigen. After antibodyincubation unbound antibody is washed away and a fluorochrome-conjugatedsecondary antibody (e.g. FITC or PE labelled antibody) which recognizesthe primary antibody is added to the solution. Unbound secondaryantibody is washed away and the cellular fluorescence is detected andquantified using the claimed aparatus. The intensity of the fluorescentsignal is proportional to the amount of the cellular antigen present.The method can be used for detection and quantification of differentcellular antigens such as HER2, EGFR, VGFR, UPR and CD moleculesincluding CD3, CD4 and CD8. In addition to antigen evaluation,information about e.g. cell number, viability and DNA content can beobtained from the same sample at the single cell level. Intracellularantigens such as the cancer markers TERT, TOP and Survivin can bedetected by a similar method by fixating and permeabilizing the cellsprior to incubation with antibody. Instead of combining the detection ofintracellular markers with measurement of viability it can be combinedwith quantification of e.g. DNA content and hence cell cycle profiling.

Materials and Methods.

CD3+ positive T cells were purified from the lymphocyte containingfraction of a Lymphoprep (Axis-Shield, #1114544) separated buffy coat bypositive selection with anti-CD3 microbeads (Miltenyi Biotec) accordingthe manufacturers protocol. Cells were then stained with primary CD3specific antibody and after 30 minutes incubation cells were washed andthen stained with R-phycoerithrin (PE) labelled secondary antibody(Antibodies from BD BioSciences). After 30 minutes incubation cells werewashed again and all viable cells were co-stained using DACM(N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide, WAKO Pure ChemicalIndustries, CAS no. 55145-14-7). The stained cells were loaded into aNC-Slide. The NC-Slide was placed in the claimed apparatus and the cellswere counted and investigated using the claimed apparatus and in-housedeveloped software.

Results.

Using the claimed apparatus to analyse cells purified usingCD3-selective microbeads we found that 95% of the cells were stainedwith the PE conjugated antibody indicating that 95% of all cellsexpressed CD3 (FIG. 25). This is in accordance with data normallyachieved by flow cytometry of cells positively selected by microbeadpurification.

It should be appreciated that reference throughout this specification to“one embodiment” or “an embodiment” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention.Therefore, it is emphasized and should be appreciated that two or morereferences to “an embodiment” or “one embodiment” or “an alternativeembodiment” in various portions of this specification are notnecessarily all referring to the same embodiment. Furthermore, theparticular features, structures or characteristics may be combined assuitable in one or more embodiments of the invention.

Throughout the foregoing description, for the purposes of explanation,numerous specific details were set forth in order to provide a thoroughunderstanding of the invention. It will be apparent, however, to oneskilled in the art that the invention may be practiced without some ofthese specific details.

Accordingly, the scope and spirit of the invention should be judged interms of the claims which follow.

1. An apparatus to illuminate a sample, said apparatus comprising: asample plane having an illumination region onto which the sample isarranged; an excitation unit having a light source to generate anexcitation light; and a lens unit, having a plurality of lenses or lenselements arranged in a two dimension arrangement, to receive theexcitation light and generate an illumination light that is directedtowards the illumination region; wherein the lens unit produces ahomogeneous illumination light to be projected on the illuminationregion of the sample plane with a high illumination efficiency.
 2. Theapparatus according to claim 1, wherein the lens unit is a micro lensarray.
 3. The apparatus according to claim 1, wherein the light sourceis selected from a dispersive light source, an emitting diode, a laserdiode and a laser.
 4. The apparatus according to claim 1, wherein thelight source includes an emittance element having an emittance area ofgreater than 0.5 mm², or greater than 1.0 mm².
 5. The apparatusaccording to claim 1, wherein the excitation unit comprises of aplurality of light sources.
 6. The apparatus according to claim 1,further comprising a collimating unit to receive the excitation lightand generate a collimated excitation light.
 7. The apparatus accordingto claim 6, wherein the collimating unit includes an array of lenses. 8.The apparatus according to claim 1, wherein the plurality of lenses orlens elements comprises at least 4 lenses.
 9. The apparatus according toclaim 1, wherein dimension of the plurality of lens or lens elements isin the range from 0.5 to 3 mm.
 10. The apparatus according to claim 1,wherein the lens unit comprises a first lens array in oppositeorientation with a second lens array.
 11. The apparatus according toclaim 1, wherein a pattern of the two dimension arrangement is similarto the shape of beam of the excitation light.
 12. The apparatusaccording to claim 1, further comprising a wavelength separation unit toreceive the illumination light and to define a wavelength band andpolarity of the illumination light.
 13. The apparatus according to claim1, further comprising a focusing unit to focus the illumination lightonto the illumination region of the sample plane.
 14. The apparatusaccording to claim 1, wherein the units are arranged on the optical axisof a photoluminescent imaging system.
 15. The apparatus according toclaim 1, wherein the units are arranged substantially off the opticalaxis of a photoluminescent imaging system.
 16. The apparatus accordingto claim 1, wherein the illumination efficiency is higher than 75%. 17.The apparatus according to claim 1, wherein the homogeneity of theillumination, defined by illumination variation in the imaged region ofthe sample material is less than 0.50.
 18. A photoluminescent imagingsystem for analysing particles in a sample, said system comprising: asample plane having an illumination region onto which the sample isarranged; an excitation unit having a light source to generate anexcitation light; and a lens unit, having a plurality of lenses or lenselements arranged in a two dimension arrangement, to receive theexcitation light and generate an illumination light that is directedtowards the illumination region; wherein the lens unit produces ahomogeneous illumination light to be projected on the illuminationregion of the sample plane with a high illumination efficiency; adetection unit comprising at least a first detector to detect signalsfrom the sample; a focusing means for focusing the signals to thedetection means; and a processor coupled to receive data from thedetector(s), wherein the system further comprises at least two differentemission filters capable of filtering signals emitted from the particlestoward the detector.
 19. A method for illuminating a sample, said methodcomprising: arranging a sample on a sample plane having an illuminationregion; generating an excitation light using an excitation unit having alight source; and generating an illumination light, directed towards theillumination region, using a lens unit that comprises a plurality oflenses or lens elements arranged in a two dimension arrangement, whereinthe lens unit produces a homogeneous illumination light to be projectedon the illumination region of the sample plane with a high illuminationefficiency.
 20. The method according to claim 19, further comprisinglabelling a particle in the sample with a fluorescent material.